RESUMO
Glutathione (GSH) is a highly abundant tripeptide thiol that performs diverse protective and biosynthetic functions in cells. While changes in GSH availability are associated with inborn errors of metabolism, cancer, and neurodegenerative disorders, studying the limiting role of GSH in physiology and disease has been challenging due to its tight regulation. To address this, we generated cell and mouse models that express a bifunctional glutathione-synthesizing enzyme from Streptococcus thermophilus (GshF), which possesses both glutamate-cysteine ligase and glutathione synthase activities. GshF expression allows efficient production of GSH in the cytosol and mitochondria and prevents cell death in response to GSH depletion, but not ferroptosis induction, indicating that GSH is not a limiting factor under lipid peroxidation. CRISPR screens using engineered enzymes further revealed genes required for cell proliferation under cellular and mitochondrial GSH depletion. Among these, we identified the glutamate-cysteine ligase modifier subunit, GCLM, as a requirement for cellular sensitivity to buthionine sulfoximine, a glutathione synthesis inhibitor. Finally, GshF expression in mice is embryonically lethal but sustains postnatal viability when restricted to adulthood. Overall, our work identifies a conditional mouse model to investigate the limiting role of GSH in physiology and disease.
Assuntos
Glutamato-Cisteína Ligase , Glutationa , Animais , Camundongos , Butionina Sulfoximina/farmacologia , Modelos Animais de Doenças , Glutamato-Cisteína Ligase/genética , Glutamato-Cisteína Ligase/metabolismo , Glutationa/metabolismo , Linhagem Celular Tumoral , HumanosRESUMO
This study was designed to discern the effect of heavy scavenger metallothionein on glutathione (GSH) deprivation-evoked cardiac anomalies and mechanisms involved with an emphasis on ferroptosis. Wild-type and cardiac metallothionein transgenic mice received GSH synthase inhibitor buthionine sulfoximine (BSO; 30 mmol/L in drinking water) for 14 days before assessment of myocardial morphology and function. BSO evoked cardiac remodeling and contractile anomalies, including cardiac hypertrophy, interstitial fibrosis, enlarged left ventricular chambers, deranged ejection fraction, fraction shortening, cardiomyocyte contractile capacity, intracellular Ca2+ handling, sarcoplasmic reticulum Ca2+ reuptake, loss of mitochondrial integrity (mitochondrial swelling, loss of aconitase activity), mitochondrial energy deficit, carbonyl damage, lipid peroxidation, ferroptosis, and apoptosis. Metallothionein itself did not affect myocardial morphology and function, although it mitigated BSO-provoked myocardial anomalies, loss of mitochondrial integrity and energy, and ferroptosis. Immunoblotting revealed down-regulated sarco(endo)plasmic reticulum Ca2+-ATPase 2a, glutathione peroxidase 4, ferroptosis-suppressing CDGSH iron-sulfur domain 1 (CISD1), and mitochondrial regulating glycogen synthase kinase-3ß phosphorylation with elevated p53, myosin heavy chain-ß isozyme, IκB phosphorylation, and solute carrier family 7 member 11 (SLC7A11) as well as unchanged SLC39A1, SLC1A5, and ferroptosis-suppressing protein 1 following BSO challenge, all of which, except glutamine transporter SLC7A11 and p53, were abrogated by metallothionein. Inhibition of CISD1 using pioglitazone nullified GSH-offered benefit against BSO-induced cardiomyocyte ferroptosis and contractile and intracellular Ca2+ derangement. Taken together, these findings support a regulatory modality for CISD1 in the impedance of ferroptosis in metallothionein-offered protection against GSH depletion-evoked cardiac aberration.
Assuntos
Cardiomiopatias , Ferroptose , Glutationa , Metalotioneína , Proteínas Mitocondriais , Animais , Camundongos , Butionina Sulfoximina/farmacologia , Cardiomiopatias/metabolismo , Cardiomiopatias/patologia , Ferroptose/efeitos dos fármacos , Glutationa/metabolismo , Metalotioneína/metabolismo , Camundongos Transgênicos , Miocárdio/metabolismo , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismoRESUMO
The effects of reduced glutathione (GSH) on skeletal muscle fatigue were investigated. GSH was depressed by buthionine sulfoximine (BSO) (100 mg/kg body wt/day) treatment for 5 days, which decreased GSH content to â¼10%. Male Wistar rats were assigned to the control (N = 18) and BSO groups (N = 17). Twelve hours after BSO treatment, the plantar flexor muscles were subjected to fatiguing stimulation (FS). Eight control and seven BSO rats were rested for 0.5 h (early stage of recovery), and the remaining were rested for 6 h (late stage of recovery). Forces were measured before FS and after rest, and physiological functions were estimated using mechanically skinned fibers. The force at 40 Hz decreased to a similar extent in both groups in the early stage of recovery and was restored in the control but not in the BSO group in the late stage of recovery. In the early stage of recovery, sarcoplasmic reticulum (SR) Ca2+ release was decreased in the control greater than in the BSO group, whereas myofibrillar Ca2+ sensitivity was increased in the control but not in the BSO group. In the late stage of recovery, SR Ca2+ release decreased and SR Ca2+ leakage increased in the BSO group but not in the control group. These results indicate that GSH depression alters the cellular mechanism of muscle fatigue in the early stage and delays force recovery in the late stage of recovery, due at least in part, to the prolonged Ca2+ leakage from the SR.
Assuntos
Depressão , Fadiga Muscular , Ratos , Masculino , Animais , Fadiga Muscular/fisiologia , Ratos Wistar , Glutationa/farmacologia , Glutationa/fisiologia , Músculo Esquelético , Butionina Sulfoximina/farmacologiaRESUMO
Buthionine sulfoximine (BSO) is a synthetic amino acid that blocks the biosynthesis of reduced glutathione (GSH), an endogenous antioxidant cellular component present in tumor cells. GSH levels have been associated with tumor cell resistance to chemotherapeutic drugs and platinum compounds. Consequently, by depleting GSH, BSO enhances the cytotoxicity of chemotherapeutic agents in drug-resistant tumors. Therefore, the aim of this study was to conduct a systematic review with meta-analysis of preclinical studies utilizing BSO in cancer treatments. The systematic search was carried out using the following databases: PubMed, Web of Science, Scopus, and EMBASE up until March 20, 2023, in order to collect preclinical studies that evaluated BSO, alone or in association, as a strategy for antineoplastic therapy. One hundred nine investigations were found to assess the cytotoxic potential of BSO alone or in combination with other compounds. Twenty-one of these met the criteria for performing the meta-analysis. The evidence gathered indicated that BSO alone exhibits cytotoxic activity. However, this compound is generally used in combination with other antineoplastic strategies, mainly chemotherapy ones, to improve cytotoxicity to carcinogenic cells and treatment efficacy. Finally, this review provides important considerations regarding BSO use in cancer treatment conditions, which might optimize future studies as a potential adjuvant antineoplastic therapeutic tool.
Assuntos
Antineoplásicos , Neoplasias , Humanos , Butionina Sulfoximina/farmacologia , Butionina Sulfoximina/uso terapêutico , Metionina Sulfoximina/uso terapêutico , Metionina Sulfoximina/toxicidade , Resistencia a Medicamentos Antineoplásicos , Neoplasias/tratamento farmacológico , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêuticoRESUMO
BACKGROUND: Emerging ferroptosis-driven therapies based on nanotechnology function either by increasing intracellular iron level or suppressing glutathione peroxidase 4 (GPX4) activity. Nevertheless, the therapeutic strategy of simultaneous iron delivery and GPX4 inhibition remains challenging and has significant scope for improvement. Moreover, current nanomedicine studies mainly use disulfide-thiol exchange to deplete glutathione (GSH) for GPX4 inactivation, which is unsatisfactory because of the compensatory effect of continuous GSH synthesis. METHODS: In this study, we design a two-in-one ferroptosis-inducing nanoplatform using iron-based metal-organic framework (MOF) that combines iron supply and GPX4 deactivation by loading the small molecule buthionine sulfoxide amine (BSO) to block de novo GSH biosynthesis, which can achieve sustainable GSH elimination and dual ferroptosis amplification. A coated lipid bilayer (L) can increase the stability of the nanoparticles and a modified tumor-homing peptide comprising arginine-glycine-aspartic acid (RGD/R) can achieve tumor-specific therapies. Moreover, as a decrease in GSH can alleviate resistance of cancer cells to chemotherapy drugs, oxaliplatin (OXA) was also loaded to obtain BSO&OXA@MOF-LR for enhanced cancer chemo-ferrotherapy in vivo. RESULTS: BSO&OXA@MOF-LR shows a robust tumor suppression effect and significantly improved the survival rate in 4T1 tumor xenograft mice, indicating a combined effect of dual amplified ferroptosis and GSH elimination sensitized apoptosis. CONCLUSION: BSO&OXA@MOF-LR is proven to be an efficient ferroptosis/apoptosis hybrid anti-cancer agent. This study is of great significance for the clinical development of novel drugs based on ferroptosis and apoptosis for enhanced cancer chemo-ferrotherapy.
Assuntos
Estruturas Metalorgânicas , Neoplasias , Humanos , Camundongos , Animais , Butionina Sulfoximina/farmacologia , Oxaliplatina/farmacologia , GlutationaRESUMO
Fatty liver disease has been strongly associated with a low glutathione (GSH) level in hepatocytes with increased oxidative stress, which is critically involved in the initiation and progression of the disease. The study investigated whether the GSH deficiency induced by buthionine sulfoximine (BSO), an inhibitor of γ-glutamyl cysteine synthetase, can be restored by the administration of GSH ester. We showed that mice fed a diet with cholesterol plus sodium cholate developed steatosis followed by hepatic GSH reduction. Moreover, the GSH level in the cytosol and mitochondria of steatosis plus BSO decreased than that of steatosis alone. Subsequent studies with the liver tissues and plasma of BSO plus steatosis revealed the accumulation of cholesterol in the hepatocytes, downregulating the concentration of GSH, antioxidant enzymes, and GSH metabolizing enzymes with a significant rise in reactive oxygen species (ROS), blood glucose level and plasma lipid profile. The administration of GSH ester in BSO-administered mice, prevented the depletion of GSH by upregulating the GSH concentration, antioxidant enzymes, and GSH metabolizing enzymes, followed by a reduction in ROS and plasma lipid concentration. The histopathological analysis showed a marked increase in inflammation followed by hepatocytes ballooning in BSO-induced group and steatosis control group, which was ameliorated by GSH ester administration. In conclusion, our data suggest that the restoration of GSH in the cytosol and mitochondria through the injection with GSH ester plays a principal role in maintaining the GSH level in the liver, thereby delaying the progression of fatty liver disease.
Assuntos
Antioxidantes , Hepatopatias , Ratos , Camundongos , Animais , Antioxidantes/farmacologia , Antioxidantes/metabolismo , Espécies Reativas de Oxigênio , Glutationa/metabolismo , Butionina Sulfoximina/farmacologia , Estresse Oxidativo , ColesterolRESUMO
Excessive glutathione (GSH), which is produced owing to abnormal metabolism of tumor cells, scavenges photo-induced reactive oxygen species (ROS) and consumes chemotherapeutic drugs, thereby attenuating the efficacy of photodynamic therapy and chemotherapy, respectively. Predominant strategies for GSH inhibition involve its chemical depletion, which only leads to a temporary therapeutic effect because GSH is replenished via various compensatory routes in tumor cells. Here, a versatile GSH-inhibiting nanosystem (termed PCNPs) for persistent synergistic therapy of cancer is reported. The porous skeleton of PCNPs allows easy encapsulation of buthionine sulfoximine (BSO) to sustainably suppress the biosynthesis of GSH. Thus, PCNPs not only demonstrate a prolonged release of BSO and improve drug utilization for efficient chemotherapy, but also act as an efficient photo-induced singlet oxygen radical generator that prevents the loss of ROS, thereby enhancing photodynamic therapy. In addition, the liposomal coating prevents cargo release in the blood, improves the accumulation of PCNPs at the tumor site, and promotes the cellular uptake of oxaliplatin and BSO. This strategy is applicable to ROS-based therapy and chemotherapy, which are suppressed by GSH, and may further enhance the synergistic effect of GSH-restrained therapy.
Assuntos
Neoplasias , Fotoquimioterapia , Butionina Sulfoximina/farmacologia , Glutationa/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Espécies Reativas de Oxigênio/metabolismoRESUMO
Two ternary copper(II) complexes with 2,2'-biquinoline (BQ) and with sulfonamides: sulfamethazine (SMT) or sulfaquinoxaline (SDQ) whose formulae are Cu(SMT)(BQ)Cl and Cu(SDQ)(BQ)Cl·CH3OH, in what follows SMTCu and SDQCu, respectively, induced oxidative stress by increasing ROS level from 1.0 µM and the reduction potential of the couple GSSG/GSH2. The co-treatment with L-buthionine sulfoximine (BSO), which inhibits the production of GSH, enhanced the effect of copper complexes on tumor cell viability and on oxidative damage. Both complexes generated DNA strand breaks given by-at least partially-the oxidation of pyrimidine bases, which caused the arrest of the cell cycle in the G2/M phase. These phenomena triggered processes of apoptosis proven by activation of caspase 3 and externalization of phosphatidylserine and loss of cell integrity from 1.0 µM. The combination with BSO induced a marked increase in the apoptotic population. On the other hand, an improved cell proliferation effect was observed when combining SDQCu with a radiation dose of 2 Gy from 1.0 µM or with 6 Gy from 1.5 µM. Finally, studies in multicellular spheroids demonstrated that even though copper(II) complexes did not inhibit cell invasion in collagen gels up to 48 h of treatment at the higher concentrations, multicellular resistance outperformed several drugs currently used in cancer treatment. Overall, our results reveal an antitumor effect of both complexes in monolayer and multicellular spheroids and an improvement with the addition of BSO. However, only SDQCu was the best adjuvant of ionizing radiation treatment.
Assuntos
Cobre , Neoplasias Pulmonares , Apoptose , Butionina Sulfoximina/farmacologia , Cobre/química , Cobre/farmacologia , Glutationa/metabolismo , Humanos , Pulmão , Neoplasias Pulmonares/tratamento farmacológico , Quinolinas , Radiação Ionizante , Sulfonamidas/farmacologiaRESUMO
8-Epidiosbulbin E acetate (EEA), a furan-containing diterpenoid lactone, is one of main component of Dioscorea bulbifera L. (DBL). It has been reported that EEA induces severe hepatotoxicity in mice and that its hepatotoxicity is associated with metabolic activation. The present study demonstrated that exposure to EEA (50, 100 or 200 µM) induced DNA damage, including significant DNA fragmentation, increases of tail DNA and olive tail moment, H2AX phosphorylation and PARP-1 activation, in cultured mouse primary hepatocytes. Similar observation was obtained in mice administered EEA at 50, 100 or 200 mg/kg. Pre-treatment with 10 µM ketoconazole (KTC), 200 µM vitamin C (VC), or 200 µM glutathione ethyl ester (GSH-OEt) reversed the over-production of reactive oxygen species (ROS) induced by EEA and attenuated susceptibility of hepatocytes to EEA-induced cytotoxicity and DNA damage in mouse primary hepatocytes. In contrast, pre-treatment with 1.0 mM L-buthionine sulfoximine (BSO) potentiated over-production of ROS, cytotoxicity and DNA damage induced by EEA. In summary, EEA induced DNA damage in cultured primary hepatocytes and the liver of mice. ROS, possibly along with DNA alkylation, participated in the observed DNA damage.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas , Diterpenos , Ativação Metabólica , Animais , Butionina Sulfoximina/metabolismo , Butionina Sulfoximina/farmacologia , DNA/metabolismo , Dano ao DNA , Diterpenos/farmacologia , Glutationa/metabolismo , Camundongos , Espécies Reativas de OxigênioRESUMO
Individual differences in the development of uncontrollable fear in response to traumatic stressors have been observed in clinic, but the underlying mechanisms remain unknown. In the present study we first conducted a meta-analysis of published clinical data and found that malondialdehyde, an oxidative stress biomarker, was significantly elevated in the blood of patients with fear-related anxiety disorders. We then carried out experimental study in rats subjected to fear conditioning. We showed that reestablishing redox homeostasis in basolateral amygdale (BLA) after exposure to fear stressors determined the capacity of learned fear inhibition. Intra-BLA infusion of buthionine sulfoximine (BSO) to deplete the most important endogenous antioxidant glutathione (GSH) blocked fear extinction, whereas intra-BLA infusion of dithiothreitol or N-acetylcysteine (a precursor of GSH) facilitated extinction. In electrophysiological studies conducted on transverse slices, we showed that fear stressors induced redox-dependent inhibition of NMDAR-mediated synaptic function, which was rescued by extinction learning or reducing agents. Our results reveal a novel pharmacological strategy for reversing impaired fear inhibition and highlight the role of GSH in the treatment of psychiatric disorders.
Assuntos
Acetilcisteína/farmacologia , Complexo Nuclear Basolateral da Amígdala/efeitos dos fármacos , Extinção Psicológica/efeitos dos fármacos , Medo/efeitos dos fármacos , Glutationa/metabolismo , Memória/efeitos dos fármacos , Animais , Complexo Nuclear Basolateral da Amígdala/metabolismo , Complexo Nuclear Basolateral da Amígdala/fisiologia , Butionina Sulfoximina/farmacologia , Condicionamento Clássico , Sinais (Psicologia) , Ditiotreitol/farmacologia , Glutationa/fisiologia , Homeostase/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Abundant glutathione (GSH) is a biological characteristic of lots of tumor cells. A growing number of studies are utilizing GSH depletion as an effective adjuvant therapy for tumor. However, due to the compensatory effect of intracellular GSH biosynthesis, GSH is hard to be completely exhausted and the strategy of GSH depletion remains challenging. Herein, we report an L-buthionine-sulfoximine (BSO)-based hypertoxic self-assembled peptide derivative (NSBSO) with dual functions of GSH depletion and biosynthesis inhibition for selective tumor ferroptosis and pyroptosis. The NSBSO consists of a hydrophobic self-assembled peptide motif and a hydrophilic peptide derivative containing BSO that inhibits the synthesis of GSH. NSBSO was cleaved by GSH and thus experienced a morphological transformation from nanoparticles to nanofibers. NSBSO showed GSH-dependent cytotoxicity and depletion of intracellular GSH. In 4T1 cells with medium GSH level, it depleted intracellular GSH and inactivated GSH peroxidase 4 (GPX4) and thus induced efficient ferroptosis. While in B16 cells with high GSH level, it exhausted GSH and triggered indirect increase of intracellular ROS and activation of Caspase 3 and gasdermin E, resulting in severe pyroptosis. These findings demonstrate that GSH depletion- and biosynthesis inhibition-induced ferroptosis and pyroptosis strategy would provide insights in designing GSH-exhausted medicines.
Assuntos
Ferroptose , Butionina Sulfoximina/farmacologia , Glutationa , PiroptoseRESUMO
The persistence of hepatotoxicity induced by N-acetyl-para-aminophenol (Acetaminophen or Paracetamol, abbreviated as APAP) as the most common cause of acute liver failure in the United States, despite the availability of N-acetylcysteine, illustrates the clinical relevance of additional therapeutic approaches. While human mesenchymal stem cells (MSCs) have shown protection in mouse models of liver injury, the MSCs used are generally not cleared for human use and it is unclear whether these effects are due to xenotransplantation. Here we evaluated GMP manufactured clinical grade human Wharton's Jelly mesenchymal stem cells (WJMSCs), which are currently being investigated in human clinical trials, in a mouse model of APAP hepatotoxicity in comparison to human dermal fibroblasts (HDFs) to address these issues. C57BL6J mice were treated with a moderate APAP overdose (300 mg/kg) and WJMSCs were administered 90 min later. Liver injury was evaluated at 6 and 24 h after APAP. WJMSCs treatment reduced APAP-induced liver injury at both time points unlike HDFs, which showed no protection. APAP-induced JNK activation as well as AIF and Smac release from mitochondria were prevented by WJMSCs treatment without influencing APAP bioactivation. Mechanistically, WJMSCs treatment upregulated expression of Gclc and Gclm to enhance recovery of liver GSH levels to attenuate mitochondrial dysfunction and accelerated recovery of pericentral hepatocytes to re-establish liver zonation and promote liver homeostasis. Notably, preventing GSH resynthesis with buthionine sulfoximine prevented the protective effects of WJMSCs. These data indicate that these GMP-manufactured WJMCs could be a clinically relevant therapeutic approach in the management of APAP hepatotoxicity in humans.
Assuntos
Doença Hepática Crônica Induzida por Substâncias e Drogas , Doença Hepática Induzida por Substâncias e Drogas , Células-Tronco Mesenquimais , Geleia de Wharton , Humanos , Camundongos , Animais , Acetaminofen/metabolismo , Acetilcisteína/farmacologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Doença Hepática Induzida por Substâncias e Drogas/prevenção & controle , Doença Hepática Induzida por Substâncias e Drogas/metabolismo , Butionina Sulfoximina/metabolismo , Butionina Sulfoximina/farmacologia , Fígado , Hepatócitos , Modelos Animais de Doenças , Fibroblastos , Camundongos Endogâmicos C57BLRESUMO
The combination of immune checkpoint blockade with chemotherapy is currently under investigation as a promising strategy for the treatment of triple negative breast cancer (TNBC). Tumor-associated macrophages (TAMs) are the most prominent component of the breast cancer microenvironment because they influence tumor progression and the response to therapies. Here we show that macrophages acquire an immunosuppressive phenotype and increase the expression of programmed death ligand-1 (PD-L1) when treated with reactive oxygen species (ROS) inducers such as the glutathione synthesis inhibitor, buthionine sulphoximine (BSO), and paclitaxel. Mechanistically, these agents cause accumulation of ROS that in turn activate NF-κB signaling to promote PD-L1 transcription and the release of immunosuppressive chemokines. Systemic in vivo administration of paclitaxel promotes PD-L1 accumulation on the surface of TAMS in a mouse model of TNBC, consistent with in vitro results. Combinatorial treatment with paclitaxel and an anti-mouse PD-L1 blocking antibody significantly improved the therapeutic efficacy of paclitaxel by reducing tumor burden and increasing the number of tumor-associated cytotoxic T cells. Our results provide a strong rationale for the use of anti-PD-L1 blockade in the treatment of TNBC patients. Furthermore, interrogation of chemotherapy-induced PD-L1 expression in TAMs is warranted to define appropriate patient selection in the use of PD-L1 blockade.
Assuntos
Antígeno B7-H1/metabolismo , Imunossupressores/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Animais , Antígeno B7-H1/genética , Neoplasias da Mama/metabolismo , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Quimiocinas , Tratamento Farmacológico , Feminino , Glutationa/metabolismo , Humanos , Camundongos , Paclitaxel/farmacologia , Fenótipo , RNA Mensageiro/metabolismo , Neoplasias de Mama Triplo Negativas , Microambiente Tumoral , Regulação para CimaRESUMO
Auranofin, as a thioredoxin reductase (TrxR) inhibitor, has promising anti-cancer activity in several cancer types. However, little is known about the inhibitory effect of auranofin on lung cancer cell growth. We, therefore, investigated the antigrowth effects of auranofin in various lung cancer cells with respect to cell death, reactive oxygen species (ROS), and glutathione (GSH) levels. Treatment with 0~5 µM auranofin decreased cell proliferation and induced cell death in Calu-6, A549, SK-LU-1, NCI-H460, and NCI-H1299 lung cancer cells at 24 h. In addition, 0~5 µM auranofin increased ROS levels, including O2â¢-, and depleted GSH levels in these cells. N-acetyl cysteine (NAC) prevented growth inhibition and mitochondrial membrane potential (MMP, ∆Ψm) loss in 3 and 5 µM auranofin-treated Calu-6 and A549 cells at 24 h, respectively, and decreased ROS levels and GSH depletion in these cells. In contrast, L-buthionine sulfoximine (BSO) enhanced cell death, MMP (∆Ψm) loss, ROS levels, and GSH depletion in auranofin-treated Calu-6 and A549 cells. Treatment with 3 and 5 µM auranofin induced caspase-3 activation and poly (ADP ribose) polymerase (PARP) cleavage in Calu-6 and A549 cells, respectively. Both were prevented by NAC, but enhanced by BSO. Moreover, TrxR activity was reduced in auranofin-treated Calu-6 and A549 cells. That activity was decreased by BSO, but increased by NAC. In conclusion, these findings demonstrate that auranofin-induced cell death is closely related to oxidative stress resulted from increased ROS levels and GSH depletion in lung cancer cells.
Assuntos
Antineoplásicos/farmacologia , Auranofina , Neoplasias Pulmonares , Acetilcisteína/metabolismo , Acetilcisteína/farmacologia , Apoptose , Auranofina/farmacologia , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Proliferação de Células , Glutationa/metabolismo , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Potencial da Membrana Mitocondrial , Espécies Reativas de Oxigênio/metabolismoRESUMO
The effect of endogenous oxidative stress induced by γ-glutamyl cysteinesynthetase inhibitor D,L-buthionine sulfoximine (BSO) on the functioning of hypoxia-induced factor 1α (HIF-1α) was studied on Caco-2 cells. BSO was added for 24 h in concentrations of 5, 10, 50, 100, and 500 µM. It was shown that BSO in concentrations of 10, 50, and 100 µM induced endogenous oxidative stress and increased the content of HIF-1α; this effect was regulated through nuclear factor of erythroid origin 2 (Nrf2). Activation of HIF-1α had an independent protective effect, as evidenced by the decrease in cell viability after HIF-1α inhibition under these conditions. When the concentration of BSO was increased to 500 µM the content of HIF-1α did not change, and cell viability decreased.
Assuntos
Hipóxia , Estresse Oxidativo , Butionina Sulfoximina/farmacologia , Células CACO-2 , Hipóxia Celular , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genéticaRESUMO
KEY MESSAGE: Enhanced glutathione content improves lateral root development by positively regulating the transcripts of root development genes responsive to glutathione treatment, thereby increasing the overall productivity of rice plants. Glutathione is primarily known as a cellular antioxidant molecule, but its role in lateral root development in rice plants has not been elucidated. Here, we have investigated its role in lateral root development of rice Oryza sativa L. Exogenous glutathione (GSH) promoted both the number and length of lateral roots in rice, and the GSH biosynthesis inhibitor buthionine sulfoximine (BSO) significantly reduced these parameters, compared to untreated plants. The inhibition by BSO was reversed with exogenous GSH. Transcript profiling by RNA-seq revealed that expression of the transcription factor genes DREB and ERF and the hormone-related genes AOS, LOX, JAZ, and SAUR were significantly downregulated in the BSO-treated plants and, in contrast, upregulated in plants treated with GSH and with GSH and BSO together. We generated OsGS-overexpressing transgenic plants in which the transgene is controlled by the abiotic-stress-inducible OsRab21 promoter to study the effect of endogenously increased GSH levels. In cold stress, transgenic rice plants enhanced stress tolerance and lateral root development by maintaining redox homeostasis and improving upregulating the expression of transcription factors and hormone-related genes involved in lateral root development. We observed improved root growth of OsGS-overexpressing plants in paddy fields compared to the wild-type controls. These traits may have alleviated transplanting stress during early growth in the field and accounted for the increased productivity. These results provide information and perspectives on the role of GSH in gene expression, lateral root development, and grain yield in rice.
Assuntos
Grão Comestível/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glutationa/farmacologia , Oryza/genética , Raízes de Plantas/genética , Biomassa , Western Blotting , Butionina Sulfoximina/farmacologia , Grão Comestível/crescimento & desenvolvimento , Grão Comestível/metabolismo , Perfilação da Expressão Gênica/métodos , Glutationa/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Ferroptosis is a form of cell death caused by iron-dependent lipid peroxidation. Cancer cells increase cystine uptake for the synthesis of glutathione (GSH), which is used by glutathione peroxidase 4 to reduce lipid peroxides. Here, we report that cystine deprivation in glioblastoma cells, but not inhibition of GSH synthesis by l-buthionine sulfoximine (BSO), induces ferroptosis. We found that cystine deprivation decreased the protein levels of ferritin heavy chain FTH1, whereas it was increased by BSO treatment. The lysosome inhibitor bafilomycin A1 or deletion of nuclear receptor coactivator 4 (NCOA4) inhibited cystine deprivation-induced decrease in FTH1 protein levels and cell death. In addition, cystine deprivation induced microtubule-associated protein light chain 3 (LC3)-II protein accumulation, suggesting that cystine deprivation induces ferritinophagy. BSO causes cell death when glioblastoma cells are treated with iron inducers, ferrous ammonium sulfate or hemin. On the other hand, cystine deprivation-induced degradation of FTH1 and cell death required glutamine. This study suggests that ferritinophagy, in addition to GSH depletion, plays an important role in cystine deprivation-induced ferroptosis in glioblastoma cells.
Assuntos
Neoplasias Encefálicas/patologia , Cistina/deficiência , Ferritinas/metabolismo , Glioblastoma/patologia , Glutationa/metabolismo , Ferro/metabolismo , Oxirredutases/metabolismo , Antimetabólitos Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Butionina Sulfoximina/farmacologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cistina/metabolismo , Ferritinas/genética , Ferroptose , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Humanos , Peroxidação de Lipídeos , Oxirredutases/genéticaRESUMO
Oxidative stress has been implicated in a variety of neurodegenerative disorders, such as Alzheimer's and Parkinson's disease. Astrocytes play a significant role in maintaining survival of neurons by supplying antioxidants such as glutathione (GSH) to neurons. Recently, we found that noradrenaline increased the intracellular GSH concentration in astrocytes via ß3 -adrenoceptor stimulation. These observations suggest that noradrenaline protects neurons from oxidative stress-induced death by increasing the supply of GSH from astrocytes to neurons via the stimulation of ß3 -adrenoceptor in astrocytes. In the present study, we examined the protective effect of noradrenaline against H2 O2 -induced neurotoxicity using two different mixed cultures: the mixed culture of human astrocytoma U-251 MG cells and human neuroblastoma SH-SY5Y cells, and the mouse primary cerebrum mixed culture of neurons and astrocytes. H2 O2 -induced neuronal cell death was significantly attenuated by pretreatment with noradrenaline in both mixed cultures but not in single culture of SH-SY5Y cells or in mouse cerebrum neuron-rich culture. The neuroprotective effect of noradrenaline was inhibited by SR59230A, a selective ß3 -adrenoceptor antagonist, and CL316243, a selective ß3 -adrenoceptor agonist, mimicked the neuroprotective effect of noradrenaline. DL-buthionine-[S,R]-sulfoximine, a GSH synthesis inhibitor, negated the neuroprotective effect of noradrenaline in both mixed cultures. MK571, which inhibits the export of GSH from astrocytes mediated by multidrug resistance-associated protein 1, also prevented the neuroprotective effect of noradrenaline. These results suggest that noradrenaline protects neurons against H2 O2 -induced death by increasing the supply of GSH from astrocytes via ß3 -adrenoceptor stimulation.
Assuntos
Astrócitos/efeitos dos fármacos , Glutationa/metabolismo , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Norepinefrina/farmacologia , Receptores Adrenérgicos beta 3/fisiologia , Agonistas de Receptores Adrenérgicos beta 3/farmacologia , Antagonistas de Receptores Adrenérgicos beta 3/farmacologia , Animais , Astrócitos/metabolismo , Astrocitoma , Encéfalo/citologia , Butionina Sulfoximina/farmacologia , Linhagem Celular Tumoral , Técnicas de Cocultura , Dioxóis/farmacologia , Humanos , Peróxido de Hidrogênio/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , Neuroblastoma , Estresse Oxidativo , Propanolaminas/farmacologia , Propionatos/farmacologia , Quinolinas/farmacologiaRESUMO
KEY MESSAGE: Studying RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of Digitalis lanata, we here provide direct evidence for the participation of PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes) in 5ß-cardenolide formation. Progesterone 5ß-reductases (P5ßR) are assumed to catalyze the reduction of progesterone to 5ß-pregnane-3,20-dione, which is a crucial step in the biosynthesis of the 5ß-cardenolides. P5ßRs are encoded by VEP1-like genes occurring ubiquitously in embryophytes. P5ßRs are substrate-promiscuous enone-1,4-reductases recently termed PRISEs (progesterone 5ß-reductase/iridoid synthase-like enzymes). Two PRISE genes, termed DlP5ßR1 (AY585867.1) and DlP5ßR2 (HM210089.1) were isolated from Digitalis lanata. To give experimental evidence for the participation of PRISEs in 5ß-cardenolide formation, we here established several RNAi-mediated DlP5ßR1 and DlP5ßR2 knockdown shoot culture lines of D. lanata. Cardenolide contents were lower in D. lanata P5ßR-RNAi lines than in wild-type shoots. We considered that the gene knockdowns may have had pleiotropic effects such as an increase in glutathione (GSH) which is known to inhibit cardenolide formation. GSH levels and expression of glutathione reductase (GR) were measured. Both were higher in the Dl P5ßR-RNAi lines than in the wild-type shoots. Cardenolide biosynthesis was restored by buthionine sulfoximine (BSO) treatment in Dl P5ßR2-RNAi lines but not in Dl P5ßR1-RNAi lines. Since progesterone is a precursor of cardenolides but can also act as a reactive electrophile species (RES), we here discriminated between these by comparing the effects of progesterone and methyl vinyl ketone, a small RES but not a precursor of cardenolides. To the best of our knowledge, we here demonstrated for the first time that P5ßR1 is involved in cardenolide formation. We also provide further evidence that PRISEs are also important for plants dealing with stress by detoxifying reactive electrophile species (RES).
Assuntos
Cardenolídeos/metabolismo , Digitalis/genética , Digitalis/metabolismo , Oxirredutases/genética , Proteínas de Plantas/genética , Butanonas/farmacologia , Butionina Sulfoximina/farmacologia , Digitalis/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Glutationa/farmacologia , Oxirredutases/metabolismo , Proteínas de Plantas/metabolismo , Brotos de Planta/genética , Plantas Geneticamente Modificadas , Progesterona/farmacologia , Interferência de RNA , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Oxidative stress and renal inflammation play a pivotal role in the pathogenesis of hypertension. The redox-sensitive transcription factor, nuclear factor E2-related factor 2 (Nrf2) is the master regulator of phase II antioxidant enzymes that protects against oxidative stress and inflammation. This study aimed to investigate the effect of Nrf2 inhibition on oxidative stress-associated hypertension and renal dopamine 1 receptor (D1R) dysfunction in mice. Male C57BL/6 J mice were treated with a pro-oxidant, L-buthionine sulfoximine (BSO) (10 mmol/L in drinking water), and ML385 (10 kg body weight/kg body weight/day, intraperitoneally), a novel Nrf2 inhibitor that blocks Nrf2 regulated downstream target genes expression. Mice treated with BSO exhibited oxidative stress, renal functional impairment, inflammation, and elevated blood pressure. Also, BSO treatment increased the activity of phase II antioxidant enzyme, NAD(P)H: quinone oxidoreductase-1 (NQO-1). BSO and ML385 co-treatment exhibited a robust increase in blood pressure, oxidative stress and intensified the renal function deterioration as indicated by a significant increase in serum creatinine, urinary albumin excretion rate, and albumin to creatinine ratio and decreased glomerular filtration rate (GFR). Also, BSO and ML385 co-treatment downregulated NQO-1 and significantly altered the inflammatory cytokines, IL-1ß and IL-10 levels. A D1R agonist SKF38393 failed to promote urinary sodium excretion indicating functional impairment in renal D1R. ML385 per se did not affect mean arterial pressure, GFR, and renal D1R function. Taken together, we concluded that the Nrf2 inhibition aggravated oxidative stress and inflammation by diminishing phase II antioxidant defense that deteriorates renal function and contributes to the development of hypertension in mice.