RESUMO
Since hydrogen sulfide (H2S) is an important endogenous gaseous mediator, therapeutic manipulation of H2S is promising for anticancer treatment. In this work, we develop a novel theranostic nanoplatform with H2S-specific and photocontrolled synergistic activation for imaging-guided H2S depletion and downregulation along with promoted photothermal therapy. Such a nanoplatform is fabricated by integration of a H2S-responsive molecule probe that can generate a cystathionine-ß-synthase (CBS) inhibitor AOAA and a photothermal transducer into an NIR-light-responsive container. Our nanoplatform can turn on NIR fluorescence specifically in H2S-rich cancers, guiding further laser irradiation. Furthermore, prominent conversion of photoenergy into heat guarantees special container melting with controllable AOAA release for H2S-level downregulation. This smart regulation of the endogenous H2S level amplifies the PTT therapeutic effect, successfully suppressing colorectal tumor in living mice under NIR fluorescence imaging guidance. Thus, we believe that this nanoplatform may provide a powerful tool toward H2S-concerned cancer treatment with an optimized diagnostic and therapeutic effect.
Assuntos
Neoplasias Colorretais , Regulação para Baixo , Sulfeto de Hidrogênio , Terapia Fototérmica , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/química , Animais , Terapia Fototérmica/métodos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/terapia , Neoplasias Colorretais/patologia , Humanos , Camundongos , Regulação para Baixo/efeitos dos fármacos , Cistationina beta-Sintase/metabolismo , Cistationina beta-Sintase/antagonistas & inibidores , Imagem Óptica , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Camundongos Endogâmicos BALB C , Camundongos Nus , Nanopartículas/química , Raios Infravermelhos , Linhagem Celular Tumoral , Nanomedicina Teranóstica/métodosRESUMO
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 NusRESUMO
Hydrogen sulfide (H2S), the third gas signal molecule, is associated with the modulation of various physiological and pathological processes. Recent studies have reevealed that endogenous H2S may promote proliferation, induce angiogenesis and inhibit apoptosis, thereby stimulating oncogenesis. Conversely, decreased endogenous H2S release suppresses growth of various tumors including breast cancer. This observation suggests an alternative tumor therapy strategy by inhibiting H2Sproducing enzymes to reduce the release of endogenous H2S. Breast cancer is the most common type of cancer in women. Due to the lack of approved targeted therapy, its recurrence and metastasis still affect its clinical treatment. In recent years, significant progress has been made in the control of breast cancer by using inhibitors on H2Sproducing enzymes. This review summarized the roles of endogenous H2Sproducing enzymes in breast cancer and the effects of the enzyme inhibitors on anticancer and antimetastasis, with the aim of providing new insights for the treatment of breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Sulfeto de Hidrogênio/antagonistas & inibidores , Neovascularização Patológica/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/patologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Sulfeto de Hidrogênio/metabolismo , Camundongos , Neovascularização Patológica/patologia , Transdução de Sinais/efeitos dos fármacos , Sulfurtransferases/antagonistas & inibidores , Sulfurtransferases/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Increased endogenous hydrogen sulfide (H2S) level by cystathionine ß-synthase (CBS) has been shown to closely relate tumorigenesis. H2S promotes angiogenesis, stimulates bioenergy metabolism and inhibits selective phosphatases. However, the role of CBS and H2S in chronic myeloid leukemia (CML) remains elusive. In this study, we found that CBS and H2S levels were increased in the bone marrow mononuclear cells of pediatric CML patients, as well as in the CML-derived K562 cells and CBS expression levels were correlated with different disease phases. Inhibition of CBS reduced the proliferation of the CML primary bone marrow mononuclear cells and induced growth inhibition, apoptosis, cell cycle arrest, and migration suppression in K562 cells and tumor xenografts. The knockdown of CBS expression by shRNA and inhibiting CBS activity by AOAA decreased the endogenous H2S levels, promoted mitochondrial-related apoptosis and inhibited the NF-κB-mediated gene expression. Our study suggests that inhibition of CBS induces cell apoptosis, as well as limits cell proliferation and migration, a potential target for the treatment of chronic myeloid leukemia.
Assuntos
Carcinogênese/genética , Cistationina beta-Sintase/genética , Sulfeto de Hidrogênio/metabolismo , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Animais , Apoptose/genética , Células da Medula Óssea/metabolismo , Células da Medula Óssea/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Criança , Cistationina beta-Sintase/antagonistas & inibidores , Metabolismo Energético/genética , Feminino , Xenoenxertos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Camundongos , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/genética , Neovascularização Patológica/patologiaRESUMO
Circadian clock genes are found in almost every cell that has a nucleus; they regulate the rhythmic nature of all processes that are cyclical. Among the genes controlled by the circadian clock, there are numerous factors that regulate key processes in the functioning of the cell. Disturbances in the functioning of the circadian clock are associated with numerous disorders. A recent study has shown the key role of H2S in regulating circadian rhythm. In this study, we investigated the in vitro effect of pharmacological inhibition of cystathionine-ß-synthase (CBS) and/or cystathionine-γ-lyase (CSE) on the circadian dynamics of Per2 expression in serum-shocked NIH-3T3 cells. Alternatively, Cbs and Cse were knocked down by transfection with siRNA. The 48-h treatment of serum-shocked NIH-3T3 cells with 1 mM dl-propargylglycine (PAG), a specific CSE inhibitor, significantly decreased the amplitude and baseline expression of Per2. During exposure to an effective CBS and CSE inhibitor (aminooxyacetic acid [AOAA]), the amplitude of oscillation and baseline expression of Per2 significantly increased. Incubation of NIH-3T3 cells with both inhibitors also significantly increased the amplitude and baseline expression of Per2 messenger RNA (mRNA). siCbs or siCse knockdowan significantly reduced the baseline and amplitude of oscillation of Per2. In conclusion, we showed that CBS/CSE/H2S pathway participates in the regulation of the circadian clock system. PAG and AOAA, change the general expression and dynamics of Per2 genes, but the increase of amplitude and overall Per2 mRNA level due to exposure to AOAA is probably caused by factors other than CBS and CSE activity.
Assuntos
Ritmo Circadiano/efeitos dos fármacos , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina gama-Liase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Proteínas Circadianas Period/metabolismo , Soro/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Cistationina beta-Sintase/deficiência , Cistationina beta-Sintase/genética , Cistationina gama-Liase/deficiência , Cistationina gama-Liase/genética , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Camundongos , Células NIH 3T3 , RNA Interferente Pequeno/genéticaRESUMO
Hydrogen sulfide (H2S) is constitutively generated in the human body and works as a gasotransmitter in synaptic transmission. In this study, we aimed to evaluate the roles of endogenous H2S in generating eupnea at the respiratory center. We employed an in situ arterially perfused preparation of decerebrated rats and recorded the central respiratory outputs. When the H2S-producing enzyme cystathionine ß-synthase (CBS) was inhibited, respiration switched from the 3-phase eupneic pattern, which consists of inspiration, postinspiration, and expiration, to gasping-like respiration, which consists of inspiration only. On the other hand, when H2S synthesis was inhibited via cystathionine γ-lyase (CSE) or when H2S synthesis was activated via CBS, eupnea remained unchanged. These results suggest that H2S produced by CBS has crucial roles in maintaining the neuronal network to generate eupnea. The mechanism of respiratory pattern generation might be switched from a network-based system to a pacemaker cell-based system in low H2S conditions.
Assuntos
Sulfeto de Hidrogênio/metabolismo , Centro Respiratório/irrigação sanguínea , Centro Respiratório/metabolismo , Animais , Seio Carotídeo/efeitos dos fármacos , Seio Carotídeo/inervação , Seio Carotídeo/metabolismo , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/metabolismo , Denervação , Ratos , Respiração , Centro Respiratório/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Canais de Sódio/metabolismoRESUMO
BACKGROUND: Hydrogen sulfide (H2S) is an endogenous mammalian gasotransmitter. Cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST) are the principal enzymes responsible for its biogenesis. A recent yeast screen suggested that disulfiram (a well-known inhibitor of aldehyde dehydrogenase and a clinically used drug in the treatment of alcoholism) may inhibit CBS in a cell-based environment. However, prior studies have not observed any direct inhibition of CBS by disulfiram. We investigated the potential role of bioconversion of disulfiram to bis(N,N-diethyldithiocarbamate)-copper(II) complex (CuDDC) in the inhibitory effect of disulfiram on H2S production and assessed its effect in two human cell types with high CBS expression: HCT116 colon cancer cells and Down syndrome (DS) fibroblasts. METHODS: H2S production from recombinant human CBS, CSE and 3-MST was measured using the fluorescent H2S probe AzMC. Mouse liver homogenate (a rich source of CBS) was also employed to measure H2S biosynthesis. The interaction of copper with accessible protein cysteine residues was evaluated using the DTNB method. Cell proliferation and viability were measured using the BrdU and MTT methods. Cellular bioenergetics was evaluated by Extracellular Flux Analysis. RESULTS: While disulfiram did not exert any significant direct inhibitory effect on any of the H2S-producing enzymes, its metabolite, CuDDC was a potent inhibitor of CBS and CSE. The mode of its action is likely related to the complexed copper molecule. In cell-based systems, the effects of disulfiram were variable. In colon cancer cells, no significant effect of disulfiram was observed on H2S production or proliferation or viability. In contrast, in DS fibroblasts, disulfiram inhibited H2S production and improved proliferation and viability. Copper, on its own, failed to have any effects on either cell type, likely due to its low cell penetration. CuDDC inhibited H2S production in both cell types studied and exerted the functional effects that would be expected from a CBS inhibitor: inhibition of cell proliferation of cancer cells and a bell-shaped effect (stimulation of proliferation at low concentration and inhibition of these responses at higher concentration) in DS cells. Control experiments using a chemical H2S donor showed that, in addition to inhibiting CBS and CSE, part of the biological effects of CuDDC relates to a direct reaction with H2S, which occurs through its complexed copper. CONCLUSIONS: Disulfiram, via its metabolite CuDDC acts as an inhibitor of CBS and a scavenger of H2S, which, in turn, potently suppresses H2S levels in various cell types. Inhibition of H2S biosynthesis may explain some of the previously reported actions of disulfiram and CuDDC in vitro and in vivo. Disulfiram or CuDDC may be considered as potential agents for the experimental therapy of various pathophysiological conditions associated with H2S overproduction.
Assuntos
Inibidores de Acetaldeído Desidrogenases/farmacologia , Cobre/farmacologia , Cistationina beta-Sintase/antagonistas & inibidores , Dissulfiram/farmacologia , Ditiocarb/análogos & derivados , Compostos Organometálicos/farmacologia , Inibidores de Acetaldeído Desidrogenases/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Quelantes/metabolismo , Quelantes/farmacologia , Cobre/metabolismo , Cistationina beta-Sintase/metabolismo , Dissulfiram/metabolismo , Ditiocarb/metabolismo , Ditiocarb/farmacologia , Relação Dose-Resposta a Droga , Feminino , Células HCT116 , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Compostos Organometálicos/metabolismoRESUMO
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 SinaisRESUMO
Cystathionine ß-synthase (CBS) is a key enzyme in the production of the signaling molecule hydrogen sulfide, deregulation of which is known to contribute to a range of serious pathological states. Involvement of hydrogen sulfide in pathways of paramount importance for cellular homeostasis renders CBS a promising drug target. An in-house focused library of heteroaromatic compounds was screened for CBS modulators by the methylene blue assay and a pyrazolopyridine derivative with a promising CBS inhibitory potential was discovered. The compound activity was readily comparable to the most potent CBS inhibitor currently known, aminoacetic acid, while a promising specificity over the related cystathionine γ-lyase was identified. To rule out any possibility that the inhibitor may bind the enzyme regulatory domain due to its high structural similarity with cofactor s-adenosylmethionine, differential scanning fluorimetry was employed. A sub-scaffold search guided follow-up screening of related compounds, providing preliminary structure-activity relationships with respect to requisites for efficient CBS inhibition by this group of heterocycles. Subsequently, a hypothesis regarding the exact binding mode of the inhibitor was devised on the basis of the available structure-activity relationships (SAR) and a deep neural networks analysis and further supported by induced-fit docking calculations.
Assuntos
Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/metabolismo , Inibidores Enzimáticos/farmacologia , Sulfeto de Hidrogênio/análise , Pirazóis/farmacologia , Piridinas/farmacologia , Inibidores Enzimáticos/química , Humanos , Modelos Moleculares , Estrutura Molecular , Redes Neurais de Computação , Pirazóis/química , Piridinas/química , S-Adenosilmetionina/química , Relação Estrutura-AtividadeRESUMO
Cystathionine-ß-synthase (CBS), the first (and rate-limiting) enzyme in the transsulfuration pathway, is an important mammalian enzyme in health and disease. Its biochemical functions under physiological conditions include the metabolism of homocysteine (a cytotoxic molecule and cardiovascular risk factor) and the generation of hydrogen sulfide (H2S), a gaseous biological mediator with multiple regulatory roles in the vascular, nervous, and immune system. CBS is up-regulated in several diseases, including Down syndrome and many forms of cancer; in these conditions, the preclinical data indicate that inhibition or inactivation of CBS exerts beneficial effects. This article overviews the current information on the expression, tissue distribution, physiological roles, and biochemistry of CBS, followed by a comprehensive overview of direct and indirect approaches to inhibit the enzyme. Among the small-molecule CBS inhibitors, the review highlights the specificity and selectivity problems related to many of the commonly used "CBS inhibitors" (e.g., aminooxyacetic acid) and provides a comprehensive review of their pharmacological actions under physiological conditions and in various disease models.
Assuntos
Cistationina beta-Sintase/metabolismo , Síndrome de Down/metabolismo , Neoplasias/metabolismo , Animais , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/genética , Síndrome de Down/tratamento farmacológico , Síndrome de Down/enzimologia , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Sulfeto de Hidrogênio/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Tacrolimo/análogos & derivados , Tacrolimo/farmacologia , Tacrolimo/uso terapêuticoRESUMO
Photo-activatable carbon monoxide (CO)-releasing molecules (photoCORMs), have recently provided help to identify the salutary effects of CO in human pathophysiology. Among them notable is the ability of CO to sensitize chemotherapeutic-resistant cancer cells. Findings from our group have shown CO to mitigate drug resistance in certain cancer cells by the inhibition of cystathionine ß-synthase (CBS), a key regulator of redox homeostasis in the cell. Diminution of the antioxidant capacity of cancer cells leads to sensitization to reactive oxygen species-producing drugs like doxorubicin and paclitaxel upon cotreatment with CO as well as in mitigating the drug effects of cisplatin. We hypothesize that the development of CO delivery techniques for coadministration with existing cancer treatment regimens may ultimately improve clinical outcomes in cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Monóxido de Carbono/metabolismo , Cistationina beta-Sintase/antagonistas & inibidores , Doxorrubicina/farmacologia , Neoplasias/tratamento farmacológico , Paclitaxel/farmacologia , Cistationina beta-Sintase/metabolismo , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The role of hydrogen sulfide (H2S) is addressed in Xenopuslaevis oocytes. Three enzymes involved in H2S metabolism, cystathionine ß-synthase, cystathionine γ-lyase, and 3-mercaptopyruvate sulfurtransferase, were detected in prophase I and metaphase II-arrested oocytes and drove an acceleration of oocyte meiosis resumption when inhibited. Moreover, meiosis resumption is associated with a significant decrease in endogenous H2S. On another hand, a dose-dependent inhibition was obtained using the H2S donor, NaHS (1 and 5 mM). NaHS impaired translation. NaHS did not induce the dissociation of the components of the M-phase promoting factor (MPF), cyclin B and Cdk1, nor directly impacted the MPF activity. However, the M-phase entry induced by microinjection of metaphase II MPF-containing cytoplasm was diminished, suggesting upstream components of the MPF auto-amplification loop were sensitive to H2S. Superoxide dismutase and catalase hindered the effects of NaHS, and this sensitivity was partially dependent on the production of reactive oxygen species (ROS). In contrast to other species, no apoptosis was promoted. These results suggest a contribution of H2S signaling in the timing of amphibian oocytes meiosis resumption.
Assuntos
Sulfeto de Hidrogênio/metabolismo , Fator Promotor de Maturação/metabolismo , Meiose/efeitos dos fármacos , Oócitos/metabolismo , Sulfetos/farmacologia , Animais , Apoptose/efeitos dos fármacos , Catalase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ciclina B/metabolismo , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Citoplasma/metabolismo , Feminino , Prófase Meiótica I/efeitos dos fármacos , Metáfase/efeitos dos fármacos , Oócitos/química , Oócitos/enzimologia , Proteínas Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sulfetos/metabolismo , Sulfurtransferases/antagonistas & inibidores , Sulfurtransferases/metabolismo , Superóxido Dismutase/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis , Fosfatases cdc25/metabolismoRESUMO
BACKGROUND: Ferroptosis is an iron-dependent, lipid peroxide-mediated cell death that may be exploited to selective elimination of damaged and malignant cells. Recent studies have identified that small-molecule erastin specifically inhibits transmembrane cystine-glutamate antiporter system xc-, prevents extracellular cystine import and ultimately causes ferroptosis in certain cancer cells. In this study, we aimed to investigate the molecular mechanism underlying erastin-induced ferroptosis resistance in ovarian cancer cells. METHODS: We treated ovarian cancer cells with erastin and examined cell viability, cellular ROS and metabolites of the transsulfuration pathway. We also depleted cystathionine ß-synthase (CBS) and NRF2 to investigate the CBS and NRF2 dependency in erastin-resistant cells. RESULTS: We found that prolonged erastin treatment induced ferroptosis resistance. Upon exposure to erastin, cells gradually adapted to cystine deprivation via sustained activation of the reverse transsulfuration pathway, allowing the cells to bypass erastin insult. CBS, the biosynthetic enzyme for cysteine, was constantly upregulated and was critical for the resistance. Knockdown of CBS by RNAi in erastin-resistant cells caused ferroptotic cell death, while CBS overexpression conferred ferroptosis resistance. We determined that the antioxidant transcriptional factor, NRF2 was constitutively activated in erastin-resistant cells and NRF2 transcriptionally upregulated CBS. Genetically repression of NRF2 enhanced ferroptosis susceptibility. CONCLUSIONS: Based on these results, we concluded that constitutive activation of NRF2/CBS signalling confers erastin-induced ferroptosis resistance. This study demonstrates a new mechanism underlying ferroptosis resistance, and has implications for the therapeutic response to erastin-induced ferroptosis.
Assuntos
Cistationina beta-Sintase/antagonistas & inibidores , Fator 2 Relacionado a NF-E2/genética , Neoplasias Ovarianas/tratamento farmacológico , Piperazinas/farmacologia , Apoptose/efeitos dos fármacos , Cistationina beta-Sintase/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Ferroptose/efeitos dos fármacos , Ferroptose/genética , Humanos , Ferro/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Piperazinas/efeitos adversos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
We investigated the effects of sulforaphane (SFN), an isothiocyanate from cruciferous vegetables, in the regulation of cerebral blood flow using cranial windows in newborn pigs. SFN administered topically (10 µM-1 mM) or systemically (0.4 mg/kg ip) caused immediate and sustained dilation of pial arterioles concomitantly with elevated H2S in periarachnoid cortical cerebrospinal fluid. H2S is a potent vasodilator of cerebral arterioles. SFN is not a H2S donor but it acts via stimulating H2S generation in the brain catalyzed by cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS). CSE/CBS inhibitors propargylglycine, ß-cyano-L-alanine, and aminooxyacetic acid blocked brain H2S generation and cerebral vasodilation caused by SFN. The SFN-elicited vasodilation requires activation of potassium channels in cerebral arterioles. The inhibitors of KATP and BK channels glibenclamide, paxilline, and iberiotoxin blocked the vasodilator effects of topical and systemic SFN, supporting the concept that H2S is the mediator of the vasodilator properties of SFN in cerebral circulation. Overall, we provide first evidence that SFN is a brain permeable compound that increases cerebral blood flow via a non-genomic mechanism that is mediated via activation of CSE/CBS-catalyzed H2S formation in neurovascular cells followed by H2S-induced activation of KATP and BK channels in arteriolar smooth muscle.
Assuntos
Arteríolas/metabolismo , Circulação Cerebrovascular/efeitos dos fármacos , Sulfeto de Hidrogênio/metabolismo , Isotiocianatos/farmacologia , Canais KATP/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Músculo Liso Vascular/metabolismo , Vasodilatadores/farmacologia , Animais , Animais Recém-Nascidos , Arteríolas/efeitos dos fármacos , Encéfalo/metabolismo , Cistationina beta-Sintase/antagonistas & inibidores , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/antagonistas & inibidores , Cistationina gama-Liase/metabolismo , Inibidores Enzimáticos/farmacologia , Feminino , Isotiocianatos/antagonistas & inibidores , Canais KATP/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Alta/efeitos dos fármacos , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Sulfóxidos , SuínosRESUMO
Acquired resistance to 5-fluorouracil (5-FU) is a major barrier to benefit from chemotherapy in colon cancer patients. Hydrogen sulfide (H2S), mainly produced by cystathionine-ß-synthase (CBS), has been reported to promote the proliferation and migration of colon cancer cells. In this study, the effect of inhibiting H2S synthesis on the sensitivity of colon cancer cell lines to 5-FU was investigated. Increased expression of CBS was validated in online database and tissue microarrays. Inhibiting H2S synthesis significantly sensitized colon cancer cell lines to 5-FU both in vitro and in vivo. Decreasing H2S synthesis utilizing shRNA lentiviruses significantly reversed the acquired resistance to 5-FU. MicroRNA sequencing was performed and miR-215-5p was revealed as one of the miRNAs with most significantly altered expression levels after CBS knock down. Epiregulin (EREG) and thymidylate synthetase (TYMS) were predicted to be potential targets of miR-215-5p. Decreasing H2S synthesis significantly decreased the expression of EREG and TYMS. These results demonstrate that inhibiting H2S synthesis can reverse the acquired resistance to 5-FU in colon cancer cells.
Assuntos
Neoplasias do Colo/tratamento farmacológico , Cistationina beta-Sintase/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fluoruracila/administração & dosagem , Sulfeto de Hidrogênio/metabolismo , RNA Interferente Pequeno/administração & dosagem , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Cistationina beta-Sintase/antagonistas & inibidores , Epirregulina/genética , Fluoruracila/farmacologia , Regulação Neoplásica da Expressão Gênica , Células HT29 , Humanos , Camundongos , MicroRNAs/genética , RNA Interferente Pequeno/farmacologia , Timidilato Sintase/genética , Regulação para Cima/efeitos dos fármacosRESUMO
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/metabolismoRESUMO
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/metabolismoRESUMO
A number of factors can trigger amyotrophic lateral sclerosis (ALS), although its precise pathogenesis is still uncertain. In a previous study done by us, poisonous liquoral levels of hydrogen sulphide (H2S) in sporadic ALS patients were reported. In the same study very high concentrations of H2S in the cerebral tissues of the familial ALS (fALS) model of the SOD1G93A mouse, were measured. The objective of this study was to test whether decreasing the levels of H2S in the fALS mouse could be beneficial. Amino-oxyacetic acid (AOA)-a systemic dual inhibitor of cystathionine-ß-synthase and cystathionine-γ lyase (two key enzymes in the production of H2S)-was administered to fALS mice. AOA treatment decreased the content of H2S in the cerebral tissues, and the lifespan of female mice increased by approximately ten days, while disease progression in male mice was not affected. The histological evaluation of the spinal cord of the females revealed a significant increase in GFAP positivity and a significant decrease in IBA1 positivity. In conclusion, the results of the study indicate that, in the animal model, the inhibition of H2S production is more effective in females. The findings reinforce the need to adequately consider sex as a relevant factor in ALS.
Assuntos
Ácido Amino-Oxiacético/farmacologia , Esclerose Lateral Amiotrófica/metabolismo , Cistationina beta-Sintase/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Sulfeto de Hidrogênio/metabolismo , Ácido Amino-Oxiacético/uso terapêutico , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/genética , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Células Cultivadas , Inibidores Enzimáticos/uso terapêutico , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuroglia/efeitos dos fármacos , Fatores Sexuais , Superóxido Dismutase-1/genéticaRESUMO
Biosynthesis of hydrogen sulfide (H2S), a key signalling molecule in human (patho)physiology, is mostly accomplished by the human enzymes cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (MST). Several lines of evidence have shown a close correlation between increased H2S production and human diseases, such as several cancer types and amyotrophic lateral sclerosis. Identifying compounds selectively and potently inhibiting the human H2S-synthesizing enzymes may therefore prove beneficial for pharmacological applications. Here, the human enzymes CBS, CSE and MST were expressed and purified from Escherichia coli, and thirty-one pyridine derivatives were synthesized and screened for their ability to bind and inhibit these enzymes. Using differential scanning fluorimetry (DSF), surface plasmon resonance (SPR), circular dichroism spectropolarimetry (CD), and activity assays based on fluorimetric and colorimetric H2S detection, two compounds (C30 and C31) sharing structural similarities were found to weakly inhibit both CBS and CSE: 1 mM C30 inhibited these enzymes by approx. 50% and 40%, respectively, while 0.5 mM C31 accounted for CBS and CSE inhibition by approx. 40% and 60%, respectively. This work, while presenting a robust methodological platform for screening putative inhibitors of the human H2S-synthesizing enzymes, highlights the importance of employing complementary methodologies in compound screenings.
Assuntos
Cistationina beta-Sintase/antagonistas & inibidores , Cistationina gama-Liase/antagonistas & inibidores , Sulfeto de Hidrogênio/metabolismo , Piridinas/farmacologia , Sulfurtransferases/antagonistas & inibidores , Dicroísmo Circular , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fluorometria/métodos , Humanos , Azul de Metileno , Piridinas/química , Sulfurtransferases/metabolismo , Ressonância de Plasmônio de SuperfícieRESUMO
Cisplatin resistance remains a major impediment to effective treatment of ovarian cancer. Despite initial platinum responsiveness, thiol-containing peptides and proteins, glutathione (GSH) and metallothionein (MT), bind and inactivate cisplatin in cancer cells. Indeed, high levels of GSH and MT in ovarian cancers impart cisplatin resistance and are predictive of poor prognosis. Cystathionine ß-synthase (CBS), an enzyme involved in sulfur metabolism, is overexpressed in ovarian cancer tissues and is itself associated with cisplatin resistance. Treatment with exogenous carbon monoxide (CO), a known inhibitor of CBS, may mitigate cisplatin resistance in ovarian cancer cells by attenuation of GSH and MT levels. Using a photo-activated CO-releasing molecule (photoCORM), [Mn(CO)3(phen)(PTA)]CF3SO3 (phenâ¯=â¯1,10-phenanthroline, PTAâ¯=â¯1,3,5-triza-7-phosphaadamantane) we assessed the ability of CO to sensitize established cisplatin-resistant ovarian cancer cell lines to cisplatin. Cisplatin-resistant cells, treated with both cisplatin and CO, exhibited significantly lower cell viability and increased poly (ADP-ribose) polymerase (PARP) cleavage versus those treated with cisplatin alone. These cisplatin-resistant cell lines overexpressed CBS and had increased steady state levels of GSH and expression of nuclear MT. Both CO treatment and lentiviral-mediated silencing of CBS attenuated GSH and nuclear MT expression in cisplatin resistant cells. We have demonstrated that CO, delivered from a photoCORM, sensitizes established cisplatin-resistant cell lines to cisplatin. Furthermore, we have presented strong evidence that the effects of CO in circumventing chemotherapeutic drug resistance is at least in part mediated by the inactivation of endogenous CBS.