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1.
Pediatrics ; 145(5)2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32245805

RESUMO

Cystinuria is an autosomal recessive disorder characterized by excessive urinary excretion of cystine, resulting in recurrent cystine kidney stones, often presenting in childhood. Current treatment options for cystinuria include dietary and/or fluid measures and potassium citrate to reduce cystine excretion and/or increase solubility. Tiopronin and D-penicillamine are used in refractory cases to bind cystine in urine, albeit with serious side effects. A recent study revealed efficacy of nutritional supplement α-lipoic acid (ALA) treatment in preventing kidney stones in a mouse model of cystinuria. Here, we report 2 pediatric patients (6 and 15 years old) with cystinuria who received regular doses of ALA in addition to conventional therapy with potassium citrate. Both patients tolerated ALA without any adverse effects and had reduced frequency of symptomatic and asymptomatic kidney stones with disappearance of existing kidney stones in 1 patient after 2 months of ALA therapy. ALA treatment markedly improved laboratory markers of cystine solubility in urine with increased cystine capacity (-223 to -1 mg/L in patient 1 and +140 to +272 mg/L in patient 2) and decreased cystine supersaturation (1.7 to 0.88 in patient 1 and 0.64 to 0.48 in patient 2) without any changes in cystine excretion or urine pH. Our findings suggest that ALA improves solubility of cystine in urine and prevents stone formation in patients with cystinuria who do not respond to diet and citrate therapy.


Assuntos
Antioxidantes/uso terapêutico , Cistina/metabolismo , Cistinúria/tratamento farmacológico , Cistinúria/urina , Ácido Tióctico/uso terapêutico , Adolescente , Criança , Feminino , Humanos
2.
Nat Cell Biol ; 22(4): 476-486, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32231310

RESUMO

SLC7A11-mediated cystine uptake is critical for maintaining redox balance and cell survival. Here we show that this comes at a significant cost for cancer cells with high levels of SLC7A11. Actively importing cystine is potentially toxic due to its low solubility, forcing cancer cells with high levels of SLC7A11 (SLC7A11high) to constitutively reduce cystine to the more soluble cysteine. This presents a significant drain on the cellular NADPH pool and renders such cells dependent on the pentose phosphate pathway. Limiting glucose supply to SLC7A11high cancer cells results in marked accumulation of intracellular cystine, redox system collapse and rapid cell death, which can be rescued by treatments that prevent disulfide accumulation. We further show that inhibitors of glucose transporters selectively kill SLC7A11high cancer cells and suppress SLC7A11high tumour growth. Our results identify a coupling between SLC7A11-associated cystine metabolism and the pentose phosphate pathway, and uncover an accompanying metabolic vulnerability for therapeutic targeting in SLC7A11high cancers.


Assuntos
Sistema y+ de Transporte de Aminoácidos/genética , Carcinoma de Células Renais/genética , Cistina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Via de Pentose Fosfato/genética , Sistema y+ de Transporte de Aminoácidos/antagonistas & inibidores , Sistema y+ de Transporte de Aminoácidos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/secundário , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dissulfetos/metabolismo , Fármacos Gastrointestinais/farmacologia , Glucose/deficiência , Transportador de Glucose Tipo 1/antagonistas & inibidores , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 3/antagonistas & inibidores , Transportador de Glucose Tipo 3/genética , Transportador de Glucose Tipo 3/metabolismo , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , Humanos , Neoplasias Renais/metabolismo , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Camundongos , Camundongos Nus , Fosfogluconato Desidrogenase/genética , Fosfogluconato Desidrogenase/metabolismo , Pirazóis/farmacologia , Quinolinas/farmacologia , Estresse Fisiológico , Sulfassalazina/farmacologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Science ; 368(6486): 85-89, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32241947

RESUMO

Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC - is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC - subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.


Assuntos
Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Cisteína/deficiência , Ferroptose , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Animais , Transportador 1 de Aminoácidos Catiônicos/genética , Linhagem Celular Tumoral , Cistationina gama-Liase/administração & dosagem , Cistationina gama-Liase/farmacologia , Cistina/metabolismo , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Deleção de Genes , Humanos , Camundongos , Camundongos Mutantes
4.
J Biol Chem ; 295(5): 1350-1365, 2020 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-31914417

RESUMO

Metabolic reprogramming in cancer cells can increase their dependence on metabolic substrates such as glucose. As such, the vulnerability of cancer cells to glucose deprivation creates an attractive opportunity for therapeutic intervention. Because it is not possible to starve tumors of glucose in vivo, here we sought to identify the mechanisms in glucose deprivation-induced cancer cell death and then designed inhibitor combinations to mimic glucose deprivation-induced cell death. Using metabolomic profiling, we found that cells undergoing glucose deprivation-induced cell death exhibited dramatic accumulation of intracellular l-cysteine and its oxidized dimer, l-cystine, and depletion of the antioxidant GSH. Building on this observation, we show that glucose deprivation-induced cell death is driven not by the lack of glucose, but rather by l-cystine import. Following glucose deprivation, the import of l-cystine and its subsequent reduction to l-cysteine depleted both NADPH and GSH pools, thereby allowing toxic accumulation of reactive oxygen species. Consistent with this model, we found that the glutamate/cystine antiporter (xCT) is required for increased sensitivity to glucose deprivation. We searched for glycolytic enzymes whose expression is essential for the survival of cancer cells with high xCT expression and identified glucose transporter type 1 (GLUT1). Testing a drug combination that co-targeted GLUT1 and GSH synthesis, we found that this combination induces synthetic lethal cell death in high xCT-expressing cell lines susceptible to glucose deprivation. These results indicate that co-targeting GLUT1 and GSH synthesis may offer a potential therapeutic approach for targeting tumors dependent on glucose for survival.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Glucose/metabolismo , Neoplasias/metabolismo , Antiporters/metabolismo , Morte Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cisteína/metabolismo , Cistina/metabolismo , Dimerização , Transportador de Glucose Tipo 1/biossíntese , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Ácido Glutâmico/metabolismo , Glutationa/biossíntese , Glutationa/metabolismo , Humanos , Metaboloma/genética , NADP/metabolismo , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Medicamentos Sintéticos/farmacologia
5.
Infect Immun ; 88(3)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31843961

RESUMO

Staphylococcus aureus is a significant human pathogen due to its capacity to cause a multitude of diseases. As such, S. aureus efficiently pillages vital nutrients from the host; however, the molecular mechanisms that support sulfur acquisition during infection have not been established. One of the most abundant extracellular sulfur-containing metabolites within the host is cysteine, which acts as the major redox buffer in the blood by transitioning between reduced and oxidized (cystine) forms. We therefore hypothesized that S. aureus acquires host-derived cysteine and cystine as sources of nutrient sulfur during systemic infection. To test this hypothesis, we used the toxic cystine analogue selenocystine to initially characterize S. aureus homologues of the Bacillus subtilis cystine transporters TcyABC and TcyP. We found that genetic inactivation of both TcyA and TcyP induced selenocystine resistance. The double mutant also failed to proliferate in medium supplemented with cystine, cysteine, or N-acetyl cysteine as the sole sulfur source. However, only TcyABC was necessary for proliferation in defined medium containing homocystine as the sulfur source. Using a murine model of systemic infection, we observed tcyP-dependent competitive defects in the liver and heart, indicating that this sulfur acquisition strategy supports proliferation of S. aureus in these organs. Phylogenetic analyses identified TcyP homologues in many pathogenic species, implying that this sulfur procurement strategy is conserved. In total, this study is the first to experimentally validate sulfur acquisition systems in S. aureus and establish their importance during pathogenesis.


Assuntos
Cistina/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Infecções Estafilocócicas/metabolismo , Staphylococcus aureus/fisiologia , Enxofre/metabolismo , Animais , Camundongos
6.
PLoS One ; 14(12): e0223954, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31800572

RESUMO

BACKGROUND: Cystinosis is a rare disorder caused by recessive mutations of the CTNS gene. Current therapy decreases cystine accumulation, thus slowing organ deterioration without reversing renal Fanconi syndrome or preventing eventual need for a kidney transplant.15-20% of cystinosis patients harbour at least one nonsense mutation in CTNS, leading to premature end of translation of the transcript. Aminoglycosides have been shown to permit translational read-through but have high toxicity level, especially in the kidney and inner ear. ELX-02, a modified aminoglycoside, retains it read-through ability without the toxicity. METHODS AND FINDINGS: We ascertained the toxicity of ELX-02 in cells and in mice as well as the effect of ELX-02 on translational read-through of nonsense mutations in cystinotic mice and human cells. ELX-02 was not toxic in vitro or in vivo, and permitted read-through of nonsense mutations in cystinotic mice and human cells. CONCLUSIONS: ELX-02 has translational read-through activity and produces a functional CTNS protein, as evidenced by reduced cystine accumulation. This reduction is comparable to cysteamine treatment. ELX-02 accumulates in the kidney but neither cytotoxicity nor nephrotoxicity was observed.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/fisiologia , Aminoglicosídeos/farmacologia , Cistina/metabolismo , Cistinose/tratamento farmacológico , Lisossomos/metabolismo , Mutação , Animais , Transporte Biológico , Cistinose/metabolismo , Cistinose/patologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Biossíntese de Proteínas
7.
J Mol Model ; 25(11): 336, 2019 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-31705320

RESUMO

xCT is a sodium-independent amino acid antiporter that imports L-cystine and exports L-glutamate in a 1:1 ratio. It is a component of heterodimeric amino acid transporter system Xc- working at the cross-roads of maintaining neurological processes and regulating antioxidant defense. The transporter has 12 transmembrane domains with intracellular N- and C-termini, and like other transporter proteins can undergo various conformational changes while switching the ligand accessibilities from intracellular to extracellular site. In the present study, we generated two homology models of human xCT in two distinct conformations: inward-facing occluded state and outward-facing open state. Our results indicated the substrate translocation channel composed of transmembrane helices TMs 1, 3, 6, 8, and 10. We docked anionic L-cystine and L-glutamate within the cavities to assess the two distinct binding scenarios for xCT as antiporter. We also assessed the interactions between the ligands and transporter and observed that ligands bind to similar residues within the channel. Using MM-PBSA/MM-GBSA approach, we computed the binding energies of these ligands to different conformational states. Cystine and glutamate bind xCT with favorable binding energies, with more favorable binding observed in inward occluded state than in outward open state. We further computed the residue-wise decomposition of these binding energies and identified the residues as essential for substrate binding/permeation. Filtering the residues that form favorable energetic contributions to the ligand binding in both the states, our studies suggest T56, A60, R135, A138, V141, Y244, A247, F250, S330, L392, and R396 as critical residues for ligand binding as well as ligand transport for any conformational state adopted by xCT during its transport cycle. .Graphical Abstract.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Cistina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sequência de Aminoácidos , Aminoácidos/metabolismo , Simulação por Computador , Ácido Glutâmico/metabolismo , Humanos , Ligantes , Estrutura Secundária de Proteína , Homologia de Sequência de Aminoácidos
8.
Arch Biochem Biophys ; 674: 108114, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31557465

RESUMO

We performed computer simulations to investigate the effect of oxidation on the extracellular cystine (CYC) uptake by the xC- antiporter. The latter is important for killing of cancer cells. Specifically, applying molecular dynamics (MD) simulations we studied the transport of CYC across xCT, i.e., the light subunit of the xC- antiporter, in charge of bidirectional transport of CYC and glutamate. We considered the outward facing (OF) configuration of xCT, and to study the effect of oxidation, we modified the Cys327 residue, located in the vicinity of the extracellular milieu, to cysteic acid (CYO327). Our computational results showed that oxidation of Cys327 results in a free energy barrier for CYC translocation, thereby blocking the access of CYC to the substrate binding site of the OF system. The formation of the energy barrier was found to be due to the conformational changes in the channel. Analysis of the MD trajectories revealed that the reorganization of the side chains of the Tyr244 and CYO327 residues play a critical role in the OF channel blocking. Indeed, the calculated distance between Tyr244 and either Cys327 or CYO327 showed a narrowing of the channel after oxidation. The obtained free energy barrier for CYC translocation was found to be 33.9kJmol-1, indicating that oxidation of Cys327, by e.g., cold atmospheric plasma, is more effective in inhibiting the xC- antiporter than in the mutation of this amino acid to Ala (yielding a barrier of 32.4kJmol-1). The inhibition of the xC- antiporter may lead to Cys starvation in some cancer cells, eventually resulting in cancer cell death.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Antiporters/metabolismo , Cistina/metabolismo , Sistema y+ de Transporte de Aminoácidos/química , Antiporters/química , Transporte Biológico , Cisteína/química , Cistina/química , Humanos , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Oxirredução , Fosfatidilcolinas/química , Conformação Proteica , Termodinâmica
9.
Indian J Pathol Microbiol ; 62(3): 457-460, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31361240

RESUMO

Nephropathic cystinosis is a rare autosomal recessive lysosomal disease characterized by accumulation of pathognomonic cystine crystals in renal and other tissues of the body. Cystinosis is caused by mutant cystinosin, the cystine transport protein located in lysosomal membranes, leading to systemic deposits of cystine and resultant end organ damage. Cystinosis is rarer in Asians than Caucasians with only a handful of cases reported from India to date. Due to its extreme rarity and clinically insidious presentation in contrast to the infantile form, the diagnosis of juvenile nephropathic cystinosis is frequently delayed or overlooked. Moreover, routine processing and sectioning of paraffin embedded tissues dissolves cystine crystals, making it difficult to diagnose this condition on light microscopic examination alone, mandating electron microscopic (EM) analysis of renal biopsies for an accurate diagnosis of this condition. We describe a case of juvenile nephropathic cystinosis presenting with uveitis and photophobia in a 17-year-old Indian male, diagnosed after EM examination of the patient's renal biopsy for evaluation of nephrotic syndrome. While highlighting the diagnostic utility of EM, we describe a few histopathologic clues which can prompt inclusion of EM analysis of renal biopsies in this setting.


Assuntos
Cistinose/diagnóstico , Síndrome Nefrótica/diagnóstico , Uveíte/diagnóstico , Adolescente , Sistemas de Transporte de Aminoácidos Neutros/genética , Biópsia , Cistina/metabolismo , Cistinose/genética , Humanos , Rim/patologia , Rim/ultraestrutura , Masculino , Microscopia Eletrônica , Síndrome Nefrótica/genética , Fotofobia/etiologia , Transtornos da Visão/etiologia
10.
ACS Appl Mater Interfaces ; 11(30): 26722-26730, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31276364

RESUMO

An effective steady-state redox balance is maintained in cancer cells, allowing for protection against oxidative stress and thereby enhancing cell proliferation and tumor growth. Disruption of this redox balance would increase the cellular content of reactive oxygen species (ROS) and potentiate oxidative stress-induced cell death in tumor cells, thus representing an effective strategy for cancer treatment. Glutathione (GSH) is a major reducing agent, and its cellular levels are determined at least partly by the availability of cysteine via xCT (SLC7A11)-mediated entry of cystine into cells. We developed a nanoplatform using ZnO nanoparticles (NPs) as a carrier, loaded with salicylazosulfapyridine (SASP), and stabilized with DSPE-PEG, to form ultra-small NPs (SASP/ZnO NPs). The goal of this NP strategy is to disrupt the redox balance in cells by two mechanisms: increased generation of ROS and decreased synthesis of GSH. Such an approach would be effective in killing tumor cells. As expected, the SASP/ZnO NPs enhanced ROS production because of ZnO and impaired GSH synthesis because of SASP-induced inhibition of xCT (SLC7A11) transport function. As a consequence, treatment of tumor cells with SASP/ZnO NPs in vitro and in vivo resulted in a synergistic disruptive effect on redox balance in tumor cells and induced cell death and decreased tumor growth. This ambidextrous approach has potential in cancer therapy by combining two complementary pathways to disrupt the redox balance in tumor cells.


Assuntos
Antineoplásicos/farmacologia , Nanopartículas/química , Neoplasias/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Antineoplásicos/química , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína/química , Cistina/metabolismo , Glutationa/metabolismo , Humanos , Nanopartículas/administração & dosagem , Neoplasias/patologia , Oxirredução/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Sulfassalazina/química , Sulfassalazina/farmacologia , Óxido de Zinco/química
11.
Asia Pac J Clin Nutr ; 28(2): 341-346, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31192563

RESUMO

BACKGROUND AND OBJECTIVES: A reliable biomarker for optimal selenium (Se) intake in lactating women is not currently available. METHODS AND STUDY DESIGN: Daily dietary Se intake in lactating women was calculated from a 24-hour meal record survey for over 3 days. Se levels in plasma and breast milk were measured through inductively coupled plasma mass spectrometry. Plasma selenoprotein P 1 levels and glutathione peroxidase 3 activity were measured using an enzyme-linked immunosorbent assay. Ultra-performance liquid chromatography-tandem mass spectrometry was used to analyze proteinaceous Se species in enzymatically digested breast milk. RESULTS: Dietary Se intakes of lactating women from Liangshan, Beijing, and Enshi were 41.6±21.2 ng/d, 51.1±22.6 ng/d, and 615±178 ng/d, respectively (p<0.05). The Se levels in the blood and breast milk were significantly associated with the dietary Se intake (p<0.05). The proteinaceous Se species in breast milk were SeMet and SeCys2. The levels of SeMet in the lactating women from Liangshan, Beijing, and Enshi were 3.31±2.44 ng Se/mL, 7.34±3.70 ng Se/mL, and 8.99±9.64 ng Se/mL, while that of SeCys2 were 13.7±12.0 ng Se/mL, 35.6±20.9 ng Se/mL, and 57.4±13.2 ng Se/mL, respectively. Notably, the concentration of SeCys2, the metabolite of unstable SeCys, reached a saturation platform, whereas no similar phenomenon were found for the total Se SeMet from Secontaining proteins. CONCLUSIONS: SeCys2 in breast milk is a potential biomarker for determining the optimal Se intake in lactating women.


Assuntos
Aleitamento Materno , Cistina/análogos & derivados , Lactação/metabolismo , Estado Nutricional , Compostos Organosselênicos/metabolismo , Selênio/deficiência , Adulto , Biomarcadores/metabolismo , China , Cistina/metabolismo , Feminino , Humanos , Leite Humano , Risco , Selênio/metabolismo
12.
Food Chem ; 295: 206-213, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31174751

RESUMO

Auricularia auricular could be useful as a candidate for human selenium supplementation. This study examined the effects of exogenous Se on the growth, yield, nutritive value, and mineral accumulation of A. auricular. Selenate or selenite (0.5-40.0 µg g-1) had no effect on mycelium morphology or the yield of fruiting bodies. In some cases, they affected the accumulation of inter-elements and significantly decreased the concentrations of copper, iron, and chromium in the Se-enriched fruiting bodies compared to that with control treatments. The polysaccharide (116.5-131.6 µg g-1) and protein (105.2-113.4 µg g-1) content in Se-enriched fruiting bodies were not significantly different from those observed in the controls (polysaccharide, 114.1 µg g-1; protein, 105.6 µg g-1). Thus, A. auricular can absorb inorganic Se from the substrate and convert it to organic Se compounds (selenocystine (≥4.1%), selenomethionine (≥91.9%), and Se-methylselenocysteine (≥2.3%)).


Assuntos
Basidiomycota/efeitos dos fármacos , Basidiomycota/metabolismo , Selênio/farmacocinética , Basidiomycota/crescimento & desenvolvimento , Biofortificação , Cromo/análise , Cromo/metabolismo , Cobre/análise , Cobre/metabolismo , Cistina/análogos & derivados , Cistina/metabolismo , Carpóforos/química , Carpóforos/efeitos dos fármacos , Carpóforos/metabolismo , Proteínas Fúngicas/análise , Proteínas Fúngicas/metabolismo , Humanos , Ferro/análise , Ferro/metabolismo , Valor Nutritivo , Compostos Organosselênicos/metabolismo , Ácido Selênico/farmacologia , Ácido Selenioso/farmacologia , Selenocisteína/análogos & derivados , Selenocisteína/metabolismo , Selenometionina/metabolismo
13.
Cancer Res ; 79(15): 3877-3890, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31175120

RESUMO

Although chemoresistance remains a primary challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC), exploiting oxidative stress might offer novel therapeutic clues. Here we explored the potential of targeting cystine/glutamate exchanger (SLC7A11/xCT), which contributes to the maintenance of intracellular glutathione (GSH). Genomic disruption of xCT via CRISPR-Cas9 was achieved in two PDAC cell lines, MiaPaCa-2 and Capan-2, and xCT-KO clones were cultivated in the presence of N-acetylcysteine. Although several cystine/cysteine transporters have been identified, our findings demonstrate that, in vitro, xCT plays the major role in intracellular cysteine balance and GSH biosynthesis. As a consequence, both xCT-KO cell lines exhibited amino acid stress with activation of GCN2 and subsequent induction of ATF4, inhibition of mTORC1, proliferation arrest, and cell death. Tumor xenograft growth was delayed but not suppressed in xCT-KO cells, which indicated both the key role of xCT and also the presence of additional mechanisms for cysteine homeostasis in vivo. Moreover, rapid depletion of intracellular GSH in xCT-KO cells led to accumulation of lipid peroxides and cell swelling. These two hallmarks of ferroptotic cell death were prevented by vitamin E or iron chelation. Finally, in vitro pharmacologic inhibition of xCT by low concentrations of erastin phenocopied xCT-KO and potentiated the cytotoxic effects of both gemcitabine and cisplatin in PDAC cell lines. In conclusion, our findings strongly support that inhibition of xCT, by its dual induction of nutritional and oxidative cellular stresses, has great potential as an anticancer strategy. SIGNIFICANCE: The cystine/glutamate exchanger xCT is essential for amino acid and redox homeostasis and its inhibition has potential for anticancer therapy by inducing ferroptosis.


Assuntos
Técnicas de Ablação/métodos , Cistina/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Nutrientes/genética , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Feminino , Humanos , Camundongos , Camundongos Nus , Estresse Oxidativo
14.
Arch Biochem Biophys ; 664: 117-126, 2019 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-30738038

RESUMO

Extracellular cystine (CYC) uptake by xC- antiporter is important for the cell viability. Especially in cancer cells, the upregulation of xC- activity is observed, which protects these cells from intracellular oxidative stress. Hence, inhibition of the CYC uptake may eventually lead to cancer cell death. Up to now, the molecular level mechanism of the CYC uptake by xC- antiporter has not been studied in detail. In this study, we applied several different simulation techniques to investigate the transport of CYC through xCT, the light subunit of the xC- antiporter, which is responsible for the CYC and glutamate translocation. Specifically, we studied the permeation of CYC across three model systems, i.e., outward facing (OF), occluded (OCC) and inward facing (IF) configurations of xCT. We also investigated the effect of mutation of Cys327 to Ala within xCT, which was also studied experimentally in literature. This allowed us to qualitatively compare our computation results with experimental observations, and thus, to validate our simulations. In summary, our simulations provide a molecular level mechanism of the transport of CYC across the xC- antiporter, more specifically, which amino acid residues in the xC- antiporter play a key role in the uptake, transport and release of CYC.


Assuntos
Sistema y+ de Transporte de Aminoácidos/metabolismo , Antiporters/metabolismo , Cistina/metabolismo , Alanina/metabolismo , Substituição de Aminoácidos , Sistema y+ de Transporte de Aminoácidos/química , Arginina/metabolismo , Sítios de Ligação , Humanos , Simulação de Dinâmica Molecular , Neoplasias/metabolismo , Neoplasias/patologia , Transporte Proteico
15.
Urolithiasis ; 47(1): 57-66, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30515543

RESUMO

Cystinuria, a genetic disorder of cystine transport, is characterized by excessive excretion of cystine in the urine and recurrent cystine stones in the kidneys and, to a lesser extent, in the bladder. Males generally are more severely affected than females. The disorder may lead to chronic kidney disease in many patients. The cystine transporter (b0,+) is a heterodimer consisting of the rBAT (encoded by SLC3A1) and b0,+AT (encoded by SLC7A9) subunits joined by a disulfide bridge. The molecular basis of cystinuria is known in great detail, and this information is now being used to define genotype-phenotype correlations. Current treatments for cystinuria include increased fluid intake to increase cystine solubility and the administration of thiol drugs for more severe cases. These drugs, however, have poor patient compliance due to adverse effects. Thus, there is a need to reduce or eliminate the risks associated with therapy for cystinuria. Four mouse models for cystinuria have been described and these models provide a resource for evaluating the safety and efficacy of new therapies for cystinuria. We are evaluating a new approach for the treatment of cystine stones based on the inhibition of cystine crystal growth by cystine analogs. Our ongoing studies indicate that cystine diamides are effective in preventing cystine stone formation in the Slc3a1 knockout mouse model for cystinuria. In addition to crystal growth, crystal aggregation is required for stone formation. Male and female mice with cystinuria have comparable levels of crystalluria, but very few female mice form stones. The identification of factors that inhibit cystine crystal aggregation in female mice may provide insight into the gender difference in disease severity in patients with cystinuria.


Assuntos
Cistina/metabolismo , Cistinúria/genética , Modelos Animais de Doenças , Cálculos Renais/etiologia , Sistemas de Transporte de Aminoácidos Básicos/genética , Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Animais , Quelantes , Ensaios Clínicos como Assunto , Cistina/análogos & derivados , Cistina/uso terapêutico , Cistinúria/complicações , Cistinúria/tratamento farmacológico , Cistinúria/epidemiologia , Desenvolvimento de Medicamentos , Feminino , Humanos , Rim/metabolismo , Cálculos Renais/prevenção & controle , Masculino , Camundongos , Camundongos Knockout , Prevalência , Eliminação Renal/genética , Índice de Gravidade de Doença , Fatores Sexuais
16.
Biol Trace Elem Res ; 187(2): 553-561, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29855849

RESUMO

Selenium (Se) is an essential trace element with multiple functions that may help mitigate adverse health conditions. Cordyceps militaris is an edible mushroom with medicinal properties. The experiment was conducted under artificial cultivation, with five Se concentrations (0, 5, 10, 20, and 40 µg g-1) and three forms of Se (selenate, selenite, and selenomethionine). C. militaris can absorb inorganic from the substrate and convert it to organic Se compounds (selenocystine, selenomethionine, and an unknown species) in fruiting bodies. Compared with the control treatment, Se applications (40 µg g-1 selenate and selenite) significantly increased the Se concentration in fruiting bodies by 130.9 and 128.1 µg g-1, respectively. The biofortification with selenate and selenite did not affect fruiting body production, in some case, but did enhance the biological efficiency. Moreover, the abundance of cordycepin and adenosine increased, while the amino acid contents remained relatively stable. Meanwhile, Se-biofortified C. militaris showed effective antioxidant activities. These results suggest that Se-biofortified C. militaris fruiting bodies may enhance human and animal health when it was included as part of a healthy diet or used as Se supplements.


Assuntos
Antioxidantes/metabolismo , Biofortificação/métodos , Cordyceps/metabolismo , Compostos de Selênio/metabolismo , Selênio/metabolismo , Adenosina/metabolismo , Animais , Antioxidantes/farmacologia , Cordyceps/efeitos dos fármacos , Cistina/análogos & derivados , Cistina/metabolismo , Desoxiadenosinas/metabolismo , Carpóforos/efeitos dos fármacos , Carpóforos/metabolismo , Humanos , Compostos Organosselênicos/metabolismo , Ácido Selênico/metabolismo , Ácido Selênico/farmacologia , Ácido Selenioso/metabolismo , Ácido Selenioso/farmacologia , Selênio/farmacologia , Compostos de Selênio/farmacologia , Selenometionina/metabolismo , Selenometionina/farmacologia
17.
Pediatr Nephrol ; 34(5): 873-881, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30413946

RESUMO

BACKGROUND: Cystinosis is an ultrarare disorder caused by mutations of the cystinosin (CTNS) gene, encoding a cystine-selective efflux channel in the lysosomes of all cells of the body. Oral therapy with cysteamine reduces intralysosomal cystine accumulation and slows organ deterioration but cannot reverse renal Fanconi syndrome nor prevent the eventual need for renal transplantation. A definitive therapeutic remains elusive. About 15% of cystinosis patients worldwide carry one or more nonsense mutations that halt translation of the CTNS protein. Aminoglycosides such as geneticin (G418) can bind to the mammalian ribosome, relax translational fidelity, and permit readthrough of premature termination codons to produce full-length protein. METHODS: To ascertain whether aminoglycosides permit readthrough of the most common CTNS nonsense mutation, W138X, we studied the effect of G418 on patient fibroblasts. RESULTS: G418 treatment induced translational readthrough of CTNSW138X constructs transfected into HEK293 cells and expression of full-length endogenous CTNS protein in homozygous W138X fibroblasts. CONCLUSIONS: Reduction in intracellular cystine indicates that the CTNS protein produced is functional as a cystine transporter. Interestingly, similar effects were seen even in W138X compound heterozygotes. These studies establish proof-of-principle for the potential of aminoglycosides to treat cystinosis and possibly other monogenic diseases caused by nonsense mutations.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Cistinose/tratamento farmacológico , Fibroblastos/efeitos dos fármacos , Gentamicinas/farmacologia , Terminação Traducional da Cadeia Peptídica/efeitos dos fármacos , Códon sem Sentido , Cistina/metabolismo , Cistinose/genética , Fibroblastos/metabolismo , Vetores Genéticos/genética , Gentamicinas/uso terapêutico , Células HEK293 , Humanos , Terminação Traducional da Cadeia Peptídica/genética , Plasmídeos/genética , RNA Mensageiro/análise , Proteínas Recombinantes/genética , Transfecção
18.
Mamm Genome ; 30(1-2): 23-33, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30591971

RESUMO

The Long-Evans Agouti (LEA/Tohm) rat has recently been established as a new rat model of type 2 diabetes. The onset of diabetes mellitus was observed only in male LEA/Tohm rats; however, urinary glucose appeared before the onset of diabetes. To clarify the genetic basis of urinary glucose, we performed genetic linkage analysis using (BN × LEA) F2 intercross progeny. A recessively acting locus responsible for urinary glucose excretion (ugl) was mapped to a 7.9 Mb region of chromosome 10, which contains the cystinosin (Ctns) gene. The Ctns gene encodes the cystine transporter, which transports cystine out of the lysosome and is responsible for nephropathic cystinosis in humans. Sequence analysis identified a 13-bp deletion in the Ctns gene, leading to a truncated and loss-of-function protein, which cause cystine accumulation in various tissues. We also developed a novel congenic rat strain harboring the Ctnsugl mutation on the F344 genetic background. Phenotypic analysis of F344-Ctnsugl rats indicated that the incidence of urinary glucose was 100% in both males and females at around 40 weeks of age, and marked cystine accumulation was observed in the tissues, as well as remarkable renal lesions and cystine crystals in the lysosomes of the renal cortex. Furthermore, treatment with cysteamine depleted the cystine contents in F344-Ctnsugl rat embryonic fibroblasts. These results indicated that the F344-Ctnsugl rat provides a novel rat model of cystinosis, which allows not only a better understanding of the pathogenesis and pathophysiology of cystinosis but will also contribute to the development of new therapies.


Assuntos
Sistemas de Transporte de Aminoácidos Neutros/genética , Cistina/metabolismo , Túbulos Renais/metabolismo , Deleção de Sequência , Alelos , Sequência de Aminoácidos , Animais , Biomarcadores , Análise Mutacional de DNA , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ligação Genética , Genótipo , Túbulos Renais/patologia , Mutação , Fenótipo , Locos de Características Quantitativas , Ratos , Ratos Transgênicos
19.
Acta Biochim Pol ; 66(4): 611-618, 2019 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-31893496

RESUMO

Acute pancreatitis (AP) is a disease defined as acute or chronic inflammatory process of the pancreas characterized by premature activation of digestive enzymes within the pancreatic acinar cells and causing pancreatic auto-digestion. In mammalian tissues, H2S is synthesized endogenously from L-cysteine in regulated enzymatic pathways catalyzed by pyridoxal phosphate-dependent enzymes: cystathionine beta - synthase (CBS), gamma - cystathionase (CTH) and cysteine aminotransferase (CAT) coupled with 3-mercaptopyruvate sulfurtransferase (MPST). In the mitochondria, hydrogen sulfide is oxidized to sulfite, which is then converted to thiosulfate (sulfane sulfur-containing compound) by thiosulfate sulfurtransferase (rhodanese; TST). The activity and the expression of CBS, CTH, MPST, and TST have been determined in vivo in pancreas of control rats, rats with acute pancreatitis and sham group. Levels of low-molecular sulfur compounds such as reduced and oxidized glutathione, cysteine, cystine and cystathionine were also determined. The study showed the significant role of MPST in H2S metabolism in pancreas. Stress caused by the surgery (sham group) and AP cause a decrease in H2S production associated with a decrease of MPST activity and expression. Markedly higher level of cysteine in the AP pancreas may be caused by a reduced rate of cysteine consumption in reaction catalyzed by MPST but it can also be a sign of the processes of proteolysis occurring in the changed tissue.


Assuntos
Ductos Biliares Extra-Hepáticos/metabolismo , Sulfeto de Hidrogênio/metabolismo , Pâncreas/metabolismo , Ductos Pancreáticos/metabolismo , Pancreatite/metabolismo , Sulfurtransferases/metabolismo , Animais , Ductos Biliares Extra-Hepáticos/cirurgia , Cistationina/metabolismo , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cisteína/metabolismo , Cistina/metabolismo , Modelos Animais de Doenças , Expressão Gênica , Glutationa/metabolismo , Ligadura , Masculino , Mitocôndrias/metabolismo , Pâncreas/patologia , Ductos Pancreáticos/cirurgia , Pancreatite/genética , Pancreatite/patologia , Ratos , Ratos Endogâmicos WKY , Sulfurtransferases/genética , Tiossulfato Sulfurtransferase/genética , Tiossulfato Sulfurtransferase/metabolismo , Transaminases/genética , Transaminases/metabolismo
20.
J Microbiol Biotechnol ; 28(12): 1982-1991, 2018 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-30394045

RESUMO

Ethanol accumulation inhibited the growth of Saccharomyces cerevisiae during wine fermentation. Autophagy and the release of reactive oxygen species (ROSs) were also induced under ethanol stress. However, the relation between autophagy and ethanol stress was still unclear. In this study, expression of the autophagy genes ATG1 and ATG8 and production of ROS under ethanol treatment in yeast were measured. The results showed that ethanol stress very significantly induced expression of ATG1 and ATG8 genes and the production of peroxide hydrogen (H2O2) and superoxide anion (O2·-). Moreover, the atg1 and atg8 mutants aggregated more H2O2 and O2·- than the wild-type yeast. In addition, inhibitors of the ROS scavenging enzyme induced expression of the ATG1 and ATG8 genes by increasing the levels of H2O2 and O2·-. In contrast, glutathione (GSH) and N-acetylcystine (NAC) decreased the ATG1 and ATG8 expression by reducing H2O2 and O2·- production. Rapamycin and 3-methyladenine also caused an obvious change in autophagy levels and simultaneously altered the release of H2O2 and O2·-. Finally, inhibitors of mitochondrial electron transport chain (mtETC) increased the production of H2O2 and O2·- and also promoted expression levels of the ATG1 and ATG8 genes. In conclusion, ethanol stress induced autophagy which was regulated by H2O2 and O2·- derived from mtETC, and in turn, the autophagy contributed to the elimination H2O2 and O2·-.


Assuntos
Autofagia/efeitos dos fármacos , Etanol/farmacologia , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Cistina/análogos & derivados , Cistina/metabolismo , Fermentação , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Membranas Mitocondriais , Mutação , Oxigênio/metabolismo , Proteínas Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Estresse Psicológico , Superóxidos/metabolismo , Transcriptoma , Vinho
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