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1.
PLoS One ; 17(10): e0276579, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36269753

RESUMO

Metabolic reprogramming is now considered a hallmark of cancer cells. KRas-driven cancer cells use glutaminolysis to generate the tricarboxylic acid cycle intermediate α-ketoglutarate via a transamination reaction between glutamate and oxaloacetate. We reported previously that exogenously supplied unsaturated fatty acids could be used to synthesize phosphatidic acid-a lipid second messenger that activates both mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2). A key target of mTORC2 is Akt-a kinase that promotes survival and regulates cell metabolism. We report here that mono-unsaturated oleic acid stimulates the phosphorylation of ATP citrate lyase (ACLY) at the Akt phosphorylation site at S455 in an mTORC2 dependent manner. Inhibition of ACLY in KRas-driven cancer cells in the absence of serum resulted in loss of cell viability. We examined the impact of glutamine (Gln) deprivation in combination with inhibition of ACLY on the viability of KRas-driven cancer cells. While Gln deprivation was somewhat toxic to KRas-driven cancer cells by itself, addition of the ACLY inhibitor SB-204990 increased the loss of cell viability. However, the transaminase inhibitor aminooxyacetate was minimally toxic and the combination of SB-204990 and aminooxtacetate led to significant loss of cell viability and strong cleavage of poly-ADP ribose polymerase-indicating apoptotic cell death. This effect was not observed in MCF7 breast cancer cells that do not have a KRas mutation or in BJ-hTERT human fibroblasts which have no oncogenic mutation. These data reveal a synthetic lethality between inhibition of glutamate oxaloacetate transaminase and ACLY inhibition that is specific for KRas-driven cancer cells and the apparent metabolic reprogramming induced by activating mutations to KRas.


Assuntos
ATP Citrato (pro-S)-Liase , Glutamina , Neoplasias , Humanos , Adenosina Difosfato Ribose , Ácido Amino-Oxiacético , ATP Citrato (pro-S)-Liase/genética , ATP Citrato (pro-S)-Liase/metabolismo , Glutamatos/genética , Glutamina/antagonistas & inibidores , Glutamina/metabolismo , Ácidos Cetoglutáricos , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/genética , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Ácidos Oleicos , Oxaloacetatos , Ácidos Fosfatídicos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transaminases/genética
2.
Redox Biol ; 56: 102466, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36113340

RESUMO

Cysteine-rich angiogenic inducer 61 (CYR61, also termed CCN family member 1 or CCN1), is a matricellular protein encoded by the CYR61 gene. This protein has been implicated in the regulation of various cancer-associated processes including tumor growth, angiogenesis, tumor cell adhesion, migration, and invasion as well as the regulation of anticancer drug resistance. Hydrogen sulfide (H2S) is a gaseous endogenous biological mediator, involved in the regulation of cellular bioenergetics, angiogenesis, invasion, and chemotherapeutic resistance in several types of cancer. H2S is produced by three enzymes: cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST). The current studies were set up to investigate if CBS or 3-MST regulates CyR61 in colon cancer cells in the context of the regulation of proliferation, migration, and survival. The study mainly utilized HCT116 cells, in which two of the principal H2S-producing enzymes, CBS and 3-MST, are highly expressed. The H2S donor GYY4137 and the polysulfide donor Na2S3 activated the CyR61 promoter in a concentration-dependent fashion. Aminooxyacetic acid (AOAA), a pharmacological inhibitor of CBS as well as HMPSNE: 2-[(4-hydroxy-6- methylpyrimidin-2-yl)sulfanyl]-1-(naphthalen-1-yl)ethan-1-one, a pharmacological inhibitor of 3-MST inhibited CyR61 mRNA expression. This effect was more pronounced in response to HMPSNE than to AOAA and occurred through the modulation of S1PR via ATF1 and CREB. CyR61 was found to play an active, but relatively minor role in maintaining colon cell proliferation. HMPSNE markedly suppressed the secretion/release of CyR61 from the colon cancer cells. Moreover, HMPSNE promoted colon cancer cell apoptosis; endogenously produced CyR61 was found to counteract this effect, at least in part via RhoA activation. Taken together, we conclude that the upregulation of 3-MST in cancer cells exerts cytoprotective effects and confers the cancer cells a more aggressive phenotype - at least in part via the modulation of CyR61 expression and release.


Assuntos
Antineoplásicos , Neoplasias do Colo , Sulfeto de Hidrogênio , Ácido Amino-Oxiacético , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Cistationina , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Cisteína , Proteína Rica em Cisteína 61 , Humanos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Neovascularização Patológica , RNA Mensageiro , Sulfurtransferases
3.
Nutrients ; 14(15)2022 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-35893874

RESUMO

The compound ß-lapachone, a naturally derived naphthoquinone, has been utilized as a potent medicinal nutrient to improve health. Over the last twelve years, numerous reports have demonstrated distinct associations of ß-lapachone and NAD(P)H: quinone oxidoreductase 1 (NQO1) protein in the amelioration of various diseases. Comprehensive research of NQO1 bioactivity has clearly confirmed the tumoricidal effects of ß-lapachone action through NAD+-keresis, in which severe DNA damage from reactive oxygen species (ROS) production triggers a poly-ADP-ribose polymerase-I (PARP1) hyperactivation cascade, culminating in NAD+/ATP depletion. Here, we report a novel combination strategy with aminooxyacetic acid (AOA), an aspartate aminotransferase inhibitor that blocks the malate-aspartate shuttle (MAS) and synergistically enhances the efficacy of ß-lapachone metabolic perturbation in NQO1+ breast cancer. We evaluated metabolic turnover in MDA-MB-231 NQO1+, MDA-MB-231 NQO1-, MDA-MB-468, and T47D cancer cells by measuring the isotopic labeling of metabolites from a [U-13C]glucose tracer. We show that ß-lapachone treatment significantly hampers lactate secretion by ~85% in NQO1+ cells. Our data demonstrate that combinatorial treatment decreases citrate, glutamate, and succinate enrichment by ~14%, ~50%, and ~65%, respectively. Differences in citrate, glutamate, and succinate fractional enrichments indicate synergistic effects on central metabolism based on the coefficient of drug interaction. Metabolic modeling suggests that increased glutamine anaplerosis is protective in the case of MAS inhibition.


Assuntos
Ácido Amino-Oxiacético , Neoplasias da Mama , Naftoquinonas , Ácido Amino-Oxiacético/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Citratos , Feminino , Glutamatos/metabolismo , Humanos , NAD/metabolismo , NAD(P)H Desidrogenase (Quinona)/metabolismo , Naftoquinonas/farmacologia , Naftoquinonas/uso terapêutico , Succinatos/metabolismo
4.
Cell Mol Life Sci ; 79(8): 438, 2022 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-35864237

RESUMO

Cystathionine beta-synthase (CBS) is a pivotal enzyme of the transsulfuration pathway responsible for diverting homocysteine to the biosynthesis of cysteine and production of hydrogen sulfide (H2S). Aberrant upregulation of CBS and overproduction of H2S contribute to pathophysiology of several diseases including cancer and Down syndrome. Therefore, pharmacological CBS inhibition has emerged as a prospective therapeutic approach. Here, we characterized binding and inhibitory mechanism of aminooxyacetic acid (AOAA), the most commonly used CBS inhibitor. We found that AOAA binds CBS tighter than its respective substrates and forms a dead-end PLP-bound intermediate featuring an oxime bond. Surprisingly, serine, but not cysteine, replaced AOAA from CBS and formed an aminoacrylate reaction intermediate, which allowed for the continuation of the catalytic cycle. Indeed, serine rescued and essentially normalized the enzymatic activity of AOAA-inhibited CBS. Cellular studies confirmed that AOAA decreased H2S production and bioenergetics, while additional serine rescued CBS activity, H2S production and mitochondrial function. The crystal structure of AOAA-bound human CBS showed a lack of hydrogen bonding with residues G305 and Y308, found in the serine-bound model. Thus, AOAA-inhibited CBS could be reactivated by serine. This difference may be important in a cellular environment in multiple pathophysiological conditions and may modulate the CBS-inhibitory activity of AOAA. In addition, our results demonstrate additional complexities of using AOAA as a CBS-specific inhibitor of H2S biogenesis and point to the urgent need to develop a potent, selective and specific pharmacological CBS inhibitor.


Assuntos
Cistationina beta-Sintase , Sulfeto de Hidrogênio , Ácido Amino-Oxiacético/farmacologia , Cistationina beta-Sintase/metabolismo , Cisteína , Humanos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Serina
5.
Front Immunol ; 13: 837669, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35251031

RESUMO

Targeting T cell metabolism is an established method of immunomodulation. Following activation, T cells engage distinct metabolic programs leading to the uptake and processing of nutrients that determine cell proliferation and differentiation. Redirection of T cell fate by modulation of these metabolic programs has been shown to boost or suppress immune responses in vitro and in vivo. Using publicly available T cell transcriptomic and proteomic datasets we identified vitamin B6-dependent transaminases as key metabolic enzymes driving T cell activation and differentiation. Inhibition of vitamin B6 metabolism using the pyridoxal 5'-phosphate (PLP) inhibitor, aminoxyacetic acid (AOA), suppresses CD8+ T cell proliferation and effector differentiation in a dose-dependent manner. We show that pyridoxal phosphate phosphatase (PDXP), a negative regulator of intracellular vitamin B6 levels, is under the control of the hypoxia-inducible transcription factor (HIF1), a central driver of T cell metabolism. Furthermore, by adoptive transfer of CD8 T cells into a C57BL/6 mouse melanoma model, we demonstrate the requirement for vitamin B6-dependent enzyme activity in mediating effective anti-tumor responses. Our findings show that vitamin B6 metabolism is required for CD8+ T cell proliferation and effector differentiation in vitro and in vivo. Targeting vitamin B6 metabolism may therefore serve as an immunodulatory strategy to improve anti-tumor immunotherapy.


Assuntos
Linfócitos T CD8-Positivos , Neoplasias , Vitamina B 6 , Ácido Amino-Oxiacético/farmacologia , Animais , Linfócitos T CD8-Positivos/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/imunologia , Fosfoproteínas Fosfatases , Proteômica , Fosfato de Piridoxal/antagonistas & inibidores , Vitamina B 6/metabolismo
6.
Biomolecules ; 11(8)2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34439739

RESUMO

Upregulation of hydrogen sulfide (H2S) biosynthesis, at least in part related to the upregulation of cystathionine ß-synthetase (CBS) in cancer cells, serves as a tumor-promoting factor and has emerged as a possible molecular target for antitumor drug development. To facilitate future clinical translation, we have synthesized a variety of novel CBS-targeting, esterase-cleavable prodrugs based on the structure of the prototypical CBS inhibitor aminooxyacetic acid (AOAA). The pharmacological properties of these compounds were evaluated in cell-free assays with recombinant human CBS protein, the human colon cancer cell line HCT116, and in vivo using various tumor-bearing mice models. The prodrug YD0251 (the isopropyl ester derivative of AOAA) was selected for detailed characterization. YD0251 exhibits improved antiproliferative efficacy in cell culture models when compared to AOAA. It is up to 18 times more potent than AOAA at suppressing HCT116 tumor growth in vivo and is effective when administered to tumor-bearing mice either via subcutaneous injection or oral gavage. Patient-derived xenografts (PDTXs) with higher levels of CBS protein grew significantly larger than tumors with lower levels, and YD0251 treatment inhibited the growth of PDTXs with elevated CBS, whereas it had no significant effect on PDTXs with low CBS protein levels. The toxicity of YD0251 was assessed in mice subjected to subchronic administration of supratherapeutic doses the inhibitor; no significant alteration in circulating markers of organ injury or histopathological alterations were noted, up to 60 mg/kg/day × 5 days. In preparation to a future theranostic concept (to match CBS inhibitor therapy to high-CBS expressors), we identified a potential plasma marker of CBS-expressing tumors. Colon cancer cells produced significant levels of lanthionine, a rare metabolic intermediate of CBS-mediated H2S biosynthesis; forced expression of CBS into non-transformed epithelial cells increased lanthionine biogenesis in vitro and in vivo (measured in the urine of tumor-bearing mice). These current results may be useful to facilitate the translation of a CBS inhibition-based antitumor concept into the clinical space.


Assuntos
Ácido Amino-Oxiacético/farmacologia , Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Cistationina beta-Sintase/antagonistas & inibidores , Pró-Fármacos/farmacologia , Animais , Células HCT116 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus
7.
Anal Chim Acta ; 1176: 338767, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34399903

RESUMO

There are numerous examples of bioactive compounds containing carbonyl groups including modified proteins with oxidation of side chain of amino acid residues to aldehyde/ketone groups which are frequently considered as markers of oxidative stress. The carbonyl unit can be also distinguished as a substructure in many illegal drugs including anabolic steroids as well as cations derivatives. Based on chemoselective formation of oximes by solid phase immobilized hydroxylamine derivatives we proposed the protocol for derivatization and selective detection of carbonylated compounds in human serum albumin hydrolysate as a complex peptide mixture and of testosterone in urine samples. This allowed for the removal of the matrix and the qualitative and quantitative analysis of the derivatized analyte by LC-MS/MS (or LC-MRM). Herein we report the preparation and chemical characterization of a novel, ChemMatrix - based resin functionalized with aminooxyacetic acid (AOA). The hydroxylamine moiety in this resin is combined with a peptide linker (GRG) containing an arginine residue to enhance the ionization efficiency. Application of an isotopically labeled carbonylated peptide ((H-Leu-Val-Thr(O)-Asp-Leu-Thr-Lys [13C6,15N2]-OH and testosterone-d3 allowed us to carry out quantitative analyses of detected compounds. Our method is general and may be applied for analysis of carbonylated compounds in biological samples. Our method based on application of functionalized resin allowed to quantify the level of free testosterone in small sample (0.5 mL) of urine, while the non-derivatized testosterone from urine sample was not detected during direct LC-MRM analysis.


Assuntos
Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em Tandem , Ácido Amino-Oxiacético , Cromatografia Líquida , Humanos , Peptídeos
8.
Arch Toxicol ; 95(4): 1303-1321, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33599830

RESUMO

Exposure to the industrial solvent trichloroethylene (TCE) has been associated with adverse pregnancy outcomes in humans and decreased fetal weight in rats. TCE kidney toxicity can occur through formation of reactive metabolites via its glutathione (GSH) conjugation metabolic pathway, largely unstudied in the context of pregnancy. To investigate the contribution of the GSH conjugation pathway and oxidative stress to TCE toxicity during pregnancy, we exposed rats orally to 480 mg TCE/kg/day from gestational day (GD) 6 to GD 16 with and without N-acetyl-L-cysteine (NAC) at 200 mg/kg/day or aminooxyacetic acid (AOAA) at 20 mg/kg/day as pre/co-treatments from GD 5-16. NAC is a reactive oxygen species scavenger that modifies the GSH conjugation pathway, and AOAA is an inhibitor of cysteine conjugate ß-lyase (CCBL) in the GSH conjugation pathway. TCE decreased fetal weight, and this was prevented by AOAA but not NAC pre/co-treatment to TCE. Although AOAA inhibited CCBL activity in maternal kidney, it did not inhibit CCBL activity in maternal liver and placenta, suggesting that AOAA prevention of TCE-induced decreased fetal weight was due to CCBL activity inhibition in the kidneys but not liver or placenta. Unexpectedly, NAC pre/co-treatment with TCE, relative to TCE treatment alone, altered placental morphology consistent with delayed developmental phenotype. Immunohistochemical staining revealed that the decidua basale, relative to basal and labyrinth zones, expressed the highest abundance of CCBL1, flavin-containing monooxygenase 3, and cleaved caspase-3. Together, the findings show the differential effects of NAC and AOAA on TCE-induced pregnancy outcomes are likely attributable to TCE metabolism modulation.


Assuntos
Acetilcisteína/farmacologia , Ácido Amino-Oxiacético/farmacologia , Reprodução/efeitos dos fármacos , Tricloroetileno/toxicidade , Animais , Inibidores Enzimáticos/farmacologia , Feminino , Sequestradores de Radicais Livres/farmacologia , Glutationa/metabolismo , Masculino , Estresse Oxidativo/efeitos dos fármacos , Placenta/efeitos dos fármacos , Gravidez , Resultado da Gravidez , Ratos , Ratos Wistar , Solventes/metabolismo , Solventes/toxicidade , Tricloroetileno/metabolismo
9.
Biomed Res Int ; 2021: 1477345, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35299827

RESUMO

As a known inhibitor of pyridoxal phosphate-dependent transaminase glutamic-oxaloacetic transaminase 1 (GOT1), aminooxyacetic acid (AOAA) has been pointed out to have potential pharmacological effects in antiepileptic, anticonvulsant, antibacterial, cancer cell proliferation inhibition, and acute myocardial infarction (MI) relief. However, its role in inflammatory bowel disease (IBD) has not been reported. Through the in vivo experiment of dextran sulfate sodium- (DSS-) induced colitis in mice, it was found that AOAA significantly attenuated the symptoms, signs, and pathological changes of colitis. In addition, AOAA treatment prevented gut barrier damages by enhancing the expression of zona occludens- (ZO-) 1, occludin, claudin-1, and E-cadherin and recovering the upregulation of the most abundant intermediate filament protein (vimentin). Moreover, the release of interleukin- (IL-) 1ß, IL-6, and tumour necrosis factor- (TNF-) α was suppressed, yet the level of IL-10 was upregulated by AOAA treatment compared to the model group. Furthermore, it was shown that AOAA administration boosted M2-like phenotype and effectively reduced M1 macrophage phenotype in the lamina propria of mouse colonic epithelium. Similarly, the effect of AOAA was verified in vitro. AOAA effectively inhibited the classically activated M1 macrophage phenotype and proinflammatory cytokine (IL-1ß, TNF-α, and IL-6) expression induced by lipopolysaccharide (LPS) and promoted M2-like phenotype. Collectively, this study reveals for the first time that short-term treatment of AOAA can significantly alleviate DSS-induced acute colitis by regulating intestinal barrier function and macrophage polarization, which provides a theoretical basis for the potential use of AOAA in the treatment of IBD.


Assuntos
Ácido Amino-Oxiacético/uso terapêutico , Colite/induzido quimicamente , Colite/tratamento farmacológico , Sulfato de Dextrana/toxicidade , GABAérgicos/uso terapêutico , Substâncias Protetoras/uso terapêutico , Animais , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL
10.
J Environ Pathol Toxicol Oncol ; 39(3): 281-290, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32865918

RESUMO

Objective-To investigate cystathionine ß synthase (CBS)/hydrogen sulfide (H2S) signaling in multiple myeloma (MM) patients and to identify its effect on the proliferation of U266 cells. Methods-Bone marrow samples of 19 MM patients and 23 healthy donors were collected. qRT-PCR was performed to measure the mRNA expression levels of H2S synthases, cystathionine ß synthase, and cystathionine γ lyase. ELISA assays quantified the amount of H2S produced by the two enzymes CBS and CSE. CCK-8 experiment was used to investigate the influence of the CBS inhibitor amino oxyacetic acid and the CSE inhibitor propargylglycine on the proliferation of U266 cells. Flow cytometry and western blotting were performed to determine the effects of AOAA, PAG, and NaHS on cell cycle distribution as well as Caspase-3 and Bcl-2 expression. Results-Patients with MM had higher level of CBS compared with healthy donors. AOAA significantly inhibited cell proliferation in both a time and concentration dependent characteristic, whereas PAG does not. After 24 hours of treatment, AOAA significantly elevated the G0/G1 phase proportion of cells, and reduced the cell distribution in both S and G2/M phases, while NaHS accelerated cell cycle progression by reducing the relative number of cells in G0/G1 phase and increasing the proportion of cells in the G2/M phase. Moreover, AOAA abolished the impact of NaHS on cell cycle progression of U266 cells. AOAA treatment also led to a significant decrease in Bcl-2 expression and dramatic increase in Caspase-3 expression, though NaHS reversed these effects. Conclusion-CBS/H2S system might have a certain effect on the proliferation and apoptosis of MM cells.


Assuntos
Apoptose , Proliferação de Células , Cistationina beta-Sintase/metabolismo , Sulfeto de Hidrogênio/metabolismo , Mieloma Múltiplo/metabolismo , Adulto , Idoso , Alcinos/farmacologia , Ácido Amino-Oxiacético/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Estudos de Casos e Controles , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/patologia , Transdução de Sinais
11.
Nitric Oxide ; 104-105: 44-50, 2020 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-32891752

RESUMO

We recently reported that hydrogen sulfide (H2S) is a possible relaxation factor in the rat bladder. However, there is no available information about the roles of central H2S in the micturition reflex, so we investigated the effects of centrally administered GYY4137 (H2S donor) and AOAA (H2S synthesis inhibitor) on the micturition reflex in urethane-anesthetized (0.8 g/kg, ip) male Wistar rats. Cystometry was performed before and after the administration of GYY4137 (3 or 10 nmol/rat, icv) or AOAA (30 or 100 µg/rat, icv). In some rats, SR95531 (GABAA receptor antagonist, 0.1 nmol/rat, icv) or SCH50911 (GABAB receptor antagonist, 0.1 nmol/rat, icv) was administered 30 min before GYY4137 administration (10 nmol/rat, icv). Centrally administered GYY4137 dose-dependently prolonged the intercontraction intervals (ICI) without altering maximum voiding pressure (MVP). On the other hand, centrally administered AOAA dose-dependently shortened ICI without altering MVP. The AOAA (30 µg/rat, icv)-induced ICI shortening was reversed in the central presence of GYY4137 (10 nmol/rat, icv). Centrally pretreated SR95531 or SCH50911 significantly attenuated the GYY4137 (10 nmol/rat, icv)-induced prolongation of ICI, respectively. These findings suggest that endogenous brain H2S can inhibit the rat micturition reflex via both GABAA and GABAB receptors in the brain.


Assuntos
Encéfalo/metabolismo , Sulfeto de Hidrogênio/metabolismo , Receptores de GABA/metabolismo , Reflexo/efeitos dos fármacos , Micção/efeitos dos fármacos , Ácido Amino-Oxiacético/farmacologia , Animais , Masculino , Morfolinas/farmacologia , Contração Muscular/fisiologia , Compostos Organotiofosforados/farmacologia , Ratos Wistar , Bexiga Urinária/fisiologia
12.
Life Sci ; 261: 118348, 2020 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-32860803

RESUMO

AIMS: 3,3'-Diindolylmethane (DIM) has limited anti-cancer effects in gastric cancer. Hydrogen sulfide (H2S) plays an important role in the tumor development and therapy, cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE), two key endogenous H2S biosynthesis enzymes, can affect endogenous H2S levels and alter cancer treatment. Our main objective was to investigate whether the aminooxyacetic acid (AOAA) and DL-Propargylglycine (PAG), two specific inhibitors of CBS and CSE, could assist DIM to exert a stronger anti-cancer effects in gastric cancer BGC-823 and SGC-7901 cells. MATERIALS AND METHODS: Cell proliferation was assayed by MTT and cell colony-forming assay. Apoptosis and migration were detected by Hoechst staining and scratch test respectively. Western blot was used to evaluate the expression of proteins related to proliferation, apoptosis and migration. KEY FINDINGS: Combination of AOAA or PAG with DIM synergistically inhibited proliferation and migration, increased apoptosis in gastric cancer cells. The p38-p53 axis was also further activated by the combination of AOAA or PAG with DIM. Exogenous H2S from sodium hydrosulfide, attenuated the efficacy of DIM in cancer cells by reducing the activation level of p38-p53 axis. Taken together, AOAA or PAG inhibited the expression of endogenous H2S biosynthesis enzymes and effectively enhanced susceptibility of gastric cancer to DIM through activating p38-p53 axis. SIGNIFICANCE: The current study highlight more precise requirements for the clinical application of sulfur-containing anti-cancer drugs, and open a new way to enhance the sensitivity of DIM in chemotherapy of gastric cancer.


Assuntos
Anticarcinógenos/farmacologia , Sulfeto de Hidrogênio/antagonistas & inibidores , Indóis/farmacologia , Neoplasias Gástricas/tratamento farmacológico , Alcinos/administração & dosagem , Alcinos/farmacologia , Ácido Amino-Oxiacético/administração & dosagem , Ácido Amino-Oxiacético/farmacologia , Anticarcinógenos/administração & dosagem , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Sinergismo Farmacológico , Glicina/administração & dosagem , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Sulfeto de Hidrogênio/metabolismo , Indóis/administração & dosagem , Neoplasias Gástricas/patologia , Proteína Supressora de Tumor p53/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
13.
Curr Mol Med ; 20(8): 624-632, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32072910

RESUMO

PURPOSE: A small molecular compound, aminooxy-acetic acid (AOA), has been shown to modulate experimental autoimmune encephalomyelitis (EAE). The current study was designed to investigate whether AOA has a similar effect on the development of experimental autoimmune uveitis (EAU) and to further explore underlying mechanisms of this drug. METHODS: EAU was induced in C57BL/6J mice by immunization with interphotoreceptor retinoid-binding protein peptide 651-670 (IRBP 651-670). AOA (500µg or 750µg) or vehicle was administered by intraperitoneal injection from day 10 to 14 after EAU induction. The severity was assessed by clinical and histological scores. The integrity of the blood retinal barrier was detected with Evans Blue. Frequencies of splenic Th1, Th17 and Foxp3+ Treg cells were examined by flow cytometry. The production of cytokines was tested by ELISA. The mRNA expression of IL-17, IFN-γ and IL-10 was detected by RT-PCR. The expression of p-Stat1 and NF-κB was detected by Western Blotting. RESULTS: AOA was found to markedly inhibit the severity of EAU, as determined by clinical and histopathological examinations. AOA can relieve the leakage of blood retinal barrier (BRB). Functional studies found a decreased frequency of Th1 and Th17 cells and an increased frequency of Treg cells in EAU mice as compared with controls. Further studies showed that AOA not only downregulated the production of the proinflammatory cytokines including IFN-γ and IL-17 but also upregulated the expression of an anti-inflammatory cytokine such as IL-10, which might be caused by inhibiting the expressions of p-Stat1 and NF-κB. CONCLUSION: This study shows that AOA inhibits the severity and development of EAU by modulating the balance between regulatory and pathogenic lymphocyte subsets.


Assuntos
Ácido Amino-Oxiacético/farmacologia , Doenças Autoimunes/prevenção & controle , GABAérgicos/farmacologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Reguladores/imunologia , Uveíte/prevenção & controle , Animais , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Proteínas do Olho/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , NF-kappa B/genética , NF-kappa B/metabolismo , Proteínas de Ligação ao Retinol/imunologia , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Uveíte/etiologia , Uveíte/metabolismo , Uveíte/patologia
14.
Artigo em Inglês | MEDLINE | ID: mdl-31907178

RESUMO

Toxoplasma gondii is an obligate intracellular protozoan parasite and a successful parasitic pathogen in diverse organisms and host cell types. Hydroxylamine (HYD) and carboxymethoxylamine (CAR) have been reported as inhibitors of aspartate aminotransferases (AATs) and interfere with the proliferation in Plasmodium falciparum Therefore, AATs are suggested as drug targets against Plasmodium The T. gondii genome encodes only one predicted AAT in both T. gondii type I strain RH and type II strain PLK. However, the effects of HYD and CAR, as well as their relationship with AAT, on T. gondii remain unclear. In this study, we found that HYD and CAR impaired the lytic cycle of T. gondiiin vitro, including the inhibition of invasion or reinvasion, intracellular replication, and egress. Importantly, HYD and CAR could control acute toxoplasmosis in vivo Further studies showed that HYD and CAR could inhibit the transamination activity of rTgAAT in vitro However, our results confirmed that deficiency of AAT in both RH and PLK did not reduce the virulence in mice, although the growth ability of the parasites was affected in vitro HYD and CAR could still inhibit the growth of AAT-deficient parasites. These findings indicated that HYD and CAR inhibition of T. gondii growth and control of toxoplasmosis can occur in an AAT-independent pathway. Overall, further studies focusing on the elucidation of the mechanism of inhibition are warranted. Our study hints at new substrates of HYD and CAR as potential drug targets to inhibit T. gondii growth.


Assuntos
Ácido Amino-Oxiacético/farmacologia , Antiprotozoários/farmacologia , Aspartato Aminotransferases/genética , Hidroxilamina/farmacologia , Proteínas de Protozoários/genética , Toxoplasma/efeitos dos fármacos , Toxoplasmose/tratamento farmacológico , Animais , Aspartato Aminotransferases/deficiência , Linhagem Celular , Chlorocebus aethiops , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/parasitologia , Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Estágios do Ciclo de Vida/efeitos dos fármacos , Estágios do Ciclo de Vida/genética , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Parasitária , Proteínas de Protozoários/metabolismo , Toxoplasma/genética , Toxoplasma/crescimento & desenvolvimento , Toxoplasma/metabolismo , Toxoplasmose/parasitologia , Células Vero
15.
J Cell Mol Med ; 24(4): 2593-2609, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31930778

RESUMO

Excessive activation of pro-inflammatory M1 macrophages following acute myocardial infarction (MI) aggravates adverse cardiac remodelling and heart dysfunction. There are two break points in the tricarboxylic acid cycle of M1 macrophages, and aspartate-arginosuccinate shunt compensates them. Aminooxyacetic acid (AOAA) is an inhibitor of aspartate aminotransferase in the aspartate-arginosuccinate shunt. Previous studies showed that manipulating macrophage metabolism may control macrophage polarization and inflammatory response. In this study, we aimed to clarify the effects of AOAA on macrophage metabolism and polarization and heart function after MI. In vitro, AOAA inhibited lactic acid and glycolysis and enhanced ATP levels in classically activated M1 macrophages. Besides, AOAA restrained pro-inflammatory M1 macrophages and promoted anti-inflammatory M2 phenotype. In vivo, MI mice were treated with AOAA or saline for three consecutive days. Remarkably, AOAA administration effectively inhibited the proportion of M1 macrophages and boosted M2-like phenotype, which subsequently attenuated infarct size as well as improved post-MI cardiac function. Additionally, AOAA attenuated NLRP3-Caspase1/IL-1ß activation and decreased the release of IL-6 and TNF-α pro-inflammatory cytokines and reciprocally increased IL-10 anti-inflammatory cytokine level in both ischaemic myocardium and M1 macrophages. In conclusion, short-term AOAA treatment significantly improves cardiac function in mice with MI by balancing macrophage polarization through modulating macrophage metabolism and inhibiting NLRP3-Caspase1/IL-1ß pathway.


Assuntos
Ácido Amino-Oxiacético/farmacologia , Cardiopatias/tratamento farmacológico , Coração/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Caspase 1/metabolismo , Cardiopatias/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Remodelação Ventricular/efeitos dos fármacos
16.
Arch Biochem Biophys ; 680: 108237, 2020 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-31881188

RESUMO

BACKGROUND AND PURPOSE: Several members of the core clock mechanism are equipped with a Per-Arnt-Sim (PAS) domain through which they can bind haem [Fe(II)]. Haem is a ligand for the orphan receptors REV-ERBα/ß (NR1D1/2), which regulate circadian rhythm and metabolism. The ability to bind haem sensitises these clock components to the action of small molecule gases, including NO, CO and H2S. Studies conducted with European hamsters revealed that during winter sleep, key clock genes stop oscillating. At the same time, H2S, when administered at subtoxic concentrations, can induce a hypometabolic state in the cell. We suppose that core clock components, including the nuclear receptors REV-ERBs, neuronal PAS domain protein 2 (nPAS2) and PER2, can be H2S targets. The general objective of this study was to investigate the effect of the H2S system on the expression profile of the core clock genes in cells in vitro. EXPERIMENTAL APPROACH: We analysed the expression of Per1, Per2, Per3, Bmal1, Cry1, Cry2, Nr1d1, Nfil-3 and Dbp messenger RNA (mRNA) in serum-shocked NIH-3T3 cells treated with a slow-releasing H2S donor (GYY4137) or the cystathionine beta-synthase (CBS) inhibitor (AOAA) cultured under constant darkness and collected during 3 days in 3 h interval. KEY RESULTS AND CONCLUSIONS AND IMPLICATIONS: We found that pharmacological CBS inhibition increased the general expression and dynamics of several clock genes. On the other hand, increased H2S decreased Per2 expression. These data suggest that CBS can affect circadian clock and effect on clock-controlled transcription output.


Assuntos
Ácido Amino-Oxiacético/farmacologia , Relógios Circadianos/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano/genética , Morfolinas/farmacologia , Compostos Organotiofosforados/farmacologia , Animais , Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Células NIH 3T3
17.
Protoplasma ; 257(1): 213-227, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31410589

RESUMO

Self-incompatibility (SI) is genetically determined reproductive barrier preventing inbreeding and thereby providing the maintenance of plant species diversity. At present, active studies of molecular bases of SI mechanisms are underway. S-RNAse-based SI in Petunia hybrida L. is a self-/non-self recognition system that allows the pistil to reject self pollen and to accept non-self pollen for outcrossing. In the present work, using fluorescent methods including the TUNEL method allowed us to reveal the presence of markers of programmed cell death (PCD), such as DNA fragmentation, in growing in vivo petunia pollen tubes during the passage of the SI reaction. The results of statistical analysis reliably proved that PCD is the factor of S-RNAse-based SI. It was found that preliminary treatment before self-pollination of stigmas of petunia self-incompatible line with aminooxyacetic acid (AOA), inhibitor of ACC synthesis, led to stimulation of pollen tubes growth when the latter did not exhibit any hallmarks of PCD. These data argue in favor of assumption that ethylene controls the passage of PCD in incompatible pollen tubes in the course of S-RNAse-based SI functioning. The involvement of the hormonal regulation in SI mechanism in P. hybrida L. is the finding observed by us for the first time.


Assuntos
Aminoácidos Cíclicos/biossíntese , Ácido Amino-Oxiacético/farmacologia , Apoptose/efeitos dos fármacos , Petunia/citologia , Petunia/fisiologia , Tubo Polínico/citologia , Autoincompatibilidade em Angiospermas/efeitos dos fármacos , Fragmentação do DNA/efeitos dos fármacos , Petunia/efeitos dos fármacos , Petunia/ultraestrutura , Tubo Polínico/efeitos dos fármacos , Tubo Polínico/ultraestrutura , Ribonucleases/metabolismo
18.
Carcinogenesis ; 41(5): 656-665, 2020 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-31276576

RESUMO

Metabolic changes accompanying a step-wise malignant transformation was investigated using a syngeneic lineage of human fibroblasts. Cell immortalization was associated with minor alterations in metabolism. Consecutive loss of cell cycle inhibition in immortalized cells resulted in increased levels of oxidative phosphorylation (OXPHOS). Overexpression of the H-Ras oncoprotein produced cells forming sarcomas in athymic mice. These transformed cells exhibited increased glucose consumption, glycolysis and a further increase in OXPHOS. Because of the markedly increased OXPHOS in transformed cells, the impact of a transaminase inhibitor, aminooxyacetic acid (AOA), which decreases glutamine influx to the tricarboxylic acid (TCA) cycle, was tested. Indeed, AOA significantly decreased proliferation of malignantly transformed fibroblasts and fibrosarcoma-derived cells in vitro and in vivo. AOA also decreased proliferation of cells susceptible to malignant transformation. Metabolomic studies in normal and transformed cells indicated that, in addition to the anticipated effect on the TCA cycle, AOA decreased production of nucleotides adenosine triphosphate (ATP) and uridine monophosphate. Exogenous nucleotides partially rescued decreased proliferation of the malignant cells treated with AOA. Our data indicate that AOA blocks several metabolic pathways essential for growth of malignant cells. Therefore, OXPHOS may provide important therapeutic targets for treatment of sarcoma.


Assuntos
Transformação Celular Neoplásica/patologia , Fibroblastos/patologia , Fibrossarcoma/patologia , Regulação Neoplásica da Expressão Gênica , Genômica/métodos , Metaboloma/efeitos dos fármacos , Pele/patologia , Ácido Amino-Oxiacético/farmacologia , Animais , Apoptose , Proliferação de Células , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibrossarcoma/tratamento farmacológico , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Glicólise , Humanos , Camundongos , Camundongos Nus , Fosforilação Oxidativa , Pele/efeitos dos fármacos , Pele/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Proc Natl Acad Sci U S A ; 116(38): 18769-18771, 2019 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-31481613

RESUMO

Down syndrome (DS) is associated with significant perturbances in mitochondrial function. Here we tested the hypothesis that the suppression of mitochondrial electron transport in DS cells is due to high expression of cystathionine-ß-synthase (CBS) and subsequent overproduction of the gaseous transmitter hydrogen sulfide (H2S). Fibroblasts from DS individuals showed higher CBS expression than control cells; CBS localization was both cytosolic and mitochondrial. DS cells produced significantly more H2S and polysulfide and exhibited a profound suppression of mitochondrial electron transport, oxygen consumption, and ATP generation. DS cells also exhibited slower proliferation rates. In DS cells, pharmacological inhibition of CBS activity with aminooxyacetate or siRNA-mediated silencing of CBS normalized cellular H2S levels, restored Complex IV activity, improved mitochondrial electron transport and ATP synthesis, and restored cell proliferation. Thus, CBS-derived H2S is responsible for the suppression of mitochondrial function in DS cells. When H2S overproduction is corrected, the tonic suppression of Complex IV is lifted, and mitochondrial electron transport is restored. CBS inhibition offers a potential approach for the pharmacological correction of DS-associated mitochondrial dysfunction.


Assuntos
Cistationina beta-Sintase/metabolismo , Síndrome de Down/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Sulfeto de Hidrogênio/metabolismo , Mitocôndrias/metabolismo , Ácido Amino-Oxiacético/farmacologia , Proliferação de Células , Células Cultivadas , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/genética , Síndrome de Down/patologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Metabolismo Energético , Feminino , Fibroblastos/metabolismo , Expressão Gênica , Humanos , Mitocôndrias/enzimologia , Fosforilação Oxidativa , Consumo de Oxigênio , RNA Interferente Pequeno/genética , Sulfetos/metabolismo
20.
Med Hypotheses ; 131: 109289, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31443780

RESUMO

Mental retardation is a progressive condition in Down syndrome: intelligence starts to decline linearly within the first year. This phenomenon could be related to the overproduction of a toxic compound, hydrogen sulfide. Indeed, a gene located on chromosome 21 controls the production of cystathionine-ß-synthase, an enzyme involved in hydrogen sulfide production in the central nervous system. It has recently been demonstrated that excess cystathionine-ß-synthase levels are needed and sufficient to induce cognitive phenotypes in mouse models of Down syndrome. Thus, two therapeutic options might be used in Down syndrome patients: the use of a specific cystathionine ß-synthase inhibitor and the use of an effective antidote to reduce hydrogen sulfide toxicity. Prenatal treatment of Down syndrome fetuses is also suggested.


Assuntos
Cistationina beta-Sintase/fisiologia , Síndrome de Down/psicologia , Sulfeto de Hidrogênio/antagonistas & inibidores , Deficiência Intelectual/terapia , Ácido Amino-Oxiacético/uso terapêutico , Animais , Benserazida/uso terapêutico , Encéfalo/metabolismo , Cromossomos Humanos Par 21/genética , Cobamidas/uso terapêutico , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/genética , Modelos Animais de Doenças , Progressão da Doença , Dissulfiram/uso terapêutico , Síndrome de Down/genética , Inibidores Enzimáticos/uso terapêutico , Dosagem de Genes , Humanos , Sulfeto de Hidrogênio/metabolismo , Recém-Nascido , Deficiência Intelectual/tratamento farmacológico , Deficiência Intelectual/genética , Camundongos , Mitocôndrias/metabolismo , Ratos , Nitrito de Sódio/uso terapêutico , Especificidade da Espécie , Tiossulfatos/metabolismo
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