RESUMO
Environmental electrophiles modify thiol groups of proteins in organs, disrupting cellular functions carried out by the modified proteins and increasing the risk of various diseases. The transcription factor NF-E2-related factor 2 (Nrf2) plays a crucial role in detoxifying electrophiles by forming glutathione adducts and subsequently excreting them into extracellular spaces. Supersulfides such as cysteine persulfides (CysSSH) produced by cystathionine γ-lyase (CSE) capture environmental electrophiles through sulfur adduct formation. However, the Nrf2 and CSE contributions to blocking environmental electrophile-mediated toxicity have yet to be evaluated. Therefore, we assessed the individual and combined roles of Nrf2 and CSE in suppressing toxicity induced by environmental electrophiles using Nrf2 knockout (KO), CSE KO, and Nrf2/CSE double KO (DKO) mice. Our findings indicate that CSE/Nrf2 DKO mice are more sensitive to environmental electrophiles compared to their single KO counterparts, highlighting the distinct mechanisms through which both pathways mitigate the toxic effects of reactive electrophiles. Moreover, diverse metabolites produced by symbiotic gut bacteria in the human body are known to exert various effects on host organ functions beyond the intestinal tract. We observed reduced blood supersulfide levels in mice lacking gut microflora compared to normal mice. Furthermore, we identified intestinal bacteria belonging to the families Ruminococcaceae and Lachnospiraceae as high CysSSH-producing bacteria. This suggests that the gut microbiota serves as a source of in vivo supersulfide molecules. These findings suggest that supersulfide derived from gut bacteria may act protectively against environmental electrophilic exposure in the host.
Assuntos
Cistationina gama-Liase , Fator 2 Relacionado a NF-E2 , Humanos , Camundongos , Animais , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/farmacologia , Glutationa/metabolismo , Compostos de Sulfidrila/metabolismo , Estresse OxidativoRESUMO
Leptin is an adipokine that regulates energy balance and immune function. Peripheral leptin administration elicits prostaglandin E2-dependent fever in rats. The gasotransmitters nitric oxide (NO) and hydrogen sulfide (H2S) are also involved in lipopolysaccharide (LPS)-induced fever response. However, there is no data in the literature indicating if these gasotransmitters have a role in leptin-induced fever response. Here, we investigate the inhibition of NO and H2S enzymes neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), and cystathionine γ-lyase (CSE) in leptin-induced fever response, respectively. Selective nNOS inhibitor 7-nitroindazole (7-NI), selective iNOS inhibitor aminoguanidine (AG), and CSE inhibitor dl-propargylglycine (PAG) were administered intraperitoneally (ip). Body temperature (Tb), food intake, and body mass were recorded in fasted male rats. Leptin (0.5 mg/kg ip) induced a significant increase in Tb, whereas AG (50 mg/kg ip), 7-NI (10 mg/kg ip), or PAG (50 mg/kg ip) caused no changes in Tb. AG, 7-NI, or PAG abolished leptin increase in Tb. Our results highlight the potential involvement of iNOS, nNOS, and CSE in leptin-induced febrile response without affecting anorexic response to leptin in fasted male rats 24 h after leptin injection. Interestingly, all the inhibitors alone had the same anorexic effect induced by leptin. These findings have implications for understanding the role of NO and H2S in leptin-induced febrile response.
Assuntos
Cistationina gama-Liase , Gasotransmissores , Animais , Masculino , Ratos , Temperatura Corporal , Cistationina gama-Liase/farmacologia , Inibidores Enzimáticos/farmacologia , Febre/induzido quimicamente , Leptina , Óxido Nítrico , Óxido Nítrico Sintase/fisiologiaRESUMO
Nasopharyngeal carcinoma (NPC) is an epithelia-derived malignancy with a distinctive geographic distribution. Cystathionine γ-lyase (CSE) is involved in cancer development and progression. Nevertheless, the role of CSE in the growth of NPC is unknown. In this study, we found that CSE levels in human NPC cells were higher than those in normal nasopharyngeal cells. CSE overexpression enhanced the proliferative, migrative, and invasive abilities of NPC cells and CSE downregulation exerted reverse effects. Overexpression of CSE decreased the expressions of cytochrome C, cleaved caspase (cas)-3, cleaved cas-9, and cleaved poly-ADP-ribose polymerase, whereas CSE knockdown exhibited reverse effects. CSE overexpression decreased reactive oxygen species (ROS) levels and the expressions of phospho (p)-extracellular signal-regulated protein kinase 1/2, p-c-Jun N-terminal kinase, and p-p38, but promoted the expressions of p-phosphatidylinositol 3-kinase (PI3K), p-AKT, and p-mammalian target of rapamycin (mTOR), whereas CSE knockdown showed oppose effects. In addition, CSE overexpression promoted NPC xenograft tumor growth and CSE knockdown decreased tumor growth by modulating proliferation, angiogenesis, cell cycle, and apoptosis. Furthermore, DL-propargylglycine (an inhibitor of CSE) dose-dependently inhibited NPC cell growth via ROS-mediated mitogen-activated protein kinase (MAPK) and PI3K/AKT/mTOR pathways without significant toxicity. In conclusion, CSE could regulate the growth of NPC cells through ROS-mediated MAPK and PI3K/AKT/mTOR cascades. CSE might be a novel tumor marker for the diagnosis and prognosis of NPC. Novel donors/drugs that inhibit the expression/activity of CSE can be developed in the treatment of NPC.
Assuntos
Cistationina gama-Liase , Neoplasias Nasofaríngeas , Humanos , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/farmacologia , Carcinoma Nasofaríngeo/metabolismo , Neoplasias Nasofaríngeas/metabolismo , Fosfatidilinositol 3-Quinase , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/farmacologia , Serina-Treonina Quinases TOR/metabolismo , AnimaisRESUMO
Objective: To investigate the effect of hydrogen sulfide (H 2S) on reactive oxygen species (ROS)-mediated endoplasmic reticulum stress in myocardial injury caused by sepsis. Methods: A sepsis model was induced in Sprague-Dawley (SD) rats by cecal ligation and puncture (CLP). The rats were randomly divided into sham operation (sham) group, sepsis (CLP) group, and sepsis+sodium hydrosulfide (NaHS) (CLP+NaHS) group. The left ventricular function of the rats was observed with echocardiography and their plasma H 2S levels were measured. Lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH) levels were measured and HE staining was done to evaluate the level of myocardial oxidative stress in rats. HE staining was done to observe the morphological changes of rat myocardium, and transmission electron microscope was used to observe the ultrastructure of myocardial mitochondria. Western blot was done to examine changes in the expression of two endogenous hydrogen sulfide synthases, cystathionine γ-lyase (CSE) and 3-mercaptopyruvate sulfur transferase (3-MST), and changes in the expression of endoplasmic reticulum stress (ERS) marker proteins, including phosphorylated (p) protein kinase R-like endoplasmic reticulum kinase (p-PERK), p-eukaryotic translation initiation factor 2α (p-eIF2α), p-inositol requires enzyme 1α (IRE1α), recombinant activating transcription factor 4 (ATF4), and C/EBP homologous protein (CHOP). TUNEL staining was performed to observe the changes of cardiomyocyte apoptosis in rats. Results: Left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS) and plasma H 2S decreased in septic rats ( P<0.05). Plasma H 2S exhibited linear correlation with LVEF and LVFS ( r 2=0.62 and r 2=0.64, all P<0.05). The ROS levels were significantly elevated in rats of the CLP group. In addition, these rats showed increased level of LDH ( P<0.05), increased expression of MDA ( P<0.05), and decreased expression of GSH ( P<0.05). Inflammatory cell infiltration and cardiomyocyte edema were observed in HE staining. Transmission electron microscopic observation revealed significant mitochondrial damage, observable mitochondrial edema, and cristae structure dissolution. The Western blot results showed that the expression levels of CSE and 3-MST decreased ( P<0.05), while the ERS marker proteins, including p-PERK, p-eIF2, IRE1α, ATF4, and CHOP, were expressed at increased levels ( P<0.05). TUNEL staining showed significant increase of apoptosis in cardiomyocytes ( P<0.05). After NaHS treatment, LVEF and LVFS increased ( P<0.05) and plasma H 2S increased in septic rats ( P<0.05). Myocardial oxidative stress levels decreased. HE staining and transmission electron microscopy showed improved myocardial morphology. Mitochondrial damage was reduced and CSE and 3-MST levels were significantly increased ( P<0.05). The expression of p-PERK, p-eIF2α, p-IRE1α, and CHOP proteins decreased ( P<0.05). A decrease in cardiomyocyte apoptosis levels was observed by TUNEL staining ( P<0.05). Conclusion: H 2S reduces septic cardiomyocyte apoptosis by inhibiting ROS-mediated ERS, thereby improving myocardial dysfunction in sepsis.
Assuntos
Sulfeto de Hidrogênio , Sepse , Animais , Ratos , Fator 4 Ativador da Transcrição/farmacologia , Apoptose , Cistationina gama-Liase/farmacologia , Estresse do Retículo Endoplasmático , Endorribonucleases/farmacologia , Fator de Iniciação 2 em Eucariotos/farmacologia , Glutationa , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/uso terapêutico , Inositol/farmacologia , Lactato Desidrogenases , Malondialdeído , Proteínas Serina-Treonina Quinases , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Sepse/complicações , Sepse/tratamento farmacológico , Volume Sistólico , Sulfetos , Enxofre/farmacologia , Função Ventricular EsquerdaRESUMO
PURPOSE: This study aimed to assess the possible healing effect of combination treatment with a hydrogen sulfide (H2S) donor, sodium hydrosulfide (NaHS) plus tadalafil on partial bladder outlet obstruction (PBOO)-induced bladder dysfunction. MATERIALS AND METHODS: A total of 75 male Sprague-Dawley rats aged 10-wk and 300-350g were divided into five groups; control; PBOO; PBOO+NaHS (5.6mg/kg/day, i.p., 6-wk); PBOO+tadalafil (2mg/kg/day, oral, 6-wk) and PBOO+NaHS+tadalafil. PBOO was created by partial urethral ligation. 6 weeks after obstruction, the in vitro contractile responses of the detrusor muscle and Western blotting, H2S and malondialdehyde assay were performed in bladder tissues. RESULTS: There was an increase in bladder weight(p<0.001) and a decrease in contractile responses to KCL(p<0.001), carbachol(p<0.01), electrical field stimulation(p<0.05) and ATP (p<0.001) in the detrusor smooth muscle of obstructed rats which was normalized after the combination treatment. Cystathionine γ-lyase and cystathionine ß-synthase, and nuclear factor kappa B protein levels did not significantly differ among groups. The obstruction induced decrement in 3-mercaptopyruvate sulfur transferase protein expression(p<0.001) and H2S levels(p<0.01) as well as increment in protein expressions of neuronal nitric oxide synthase (NO, p<0.001), endothelial NOS (p<0.05), inducible NOS(p<0.001), hypoxia-inducible factor 1-alpha (p<0.01), and malondialdehyde levels (p<0.01), when combined treatment entirely normalized. CONCLUSIONS: Combination therapy has beneficial effects on bladder dysfunction via regulating both H2S and nitric oxide pathways as well as downregulation of oxidative stress and hypoxia. The synergistic effect of H2S and nitric oxide is likely to modulate bladder function, which supports the combined therapy for enhancing clinical outcomes in men with BPH/LUTS.
Assuntos
Sulfeto de Hidrogênio , Obstrução do Colo da Bexiga Urinária , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Trifosfato de Adenosina/uso terapêutico , Animais , Carbacol/metabolismo , Carbacol/farmacologia , Carbacol/uso terapêutico , Cistationina beta-Sintase/metabolismo , Cistationina beta-Sintase/farmacologia , Cistationina beta-Sintase/uso terapêutico , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/farmacologia , Cistationina gama-Liase/uso terapêutico , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/uso terapêutico , Hipóxia/tratamento farmacológico , Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/metabolismo , Fator 1 Induzível por Hipóxia/farmacologia , Fator 1 Induzível por Hipóxia/uso terapêutico , Masculino , Malondialdeído , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Sulfetos , Enxofre/metabolismo , Enxofre/farmacologia , Enxofre/uso terapêutico , Tadalafila/farmacologia , Tadalafila/uso terapêutico , Transferases/metabolismo , Transferases/farmacologia , Transferases/uso terapêutico , Bexiga Urinária , Obstrução do Colo da Bexiga Urinária/tratamento farmacológicoRESUMO
Hydrogen sulfide (H2S) has been witnessed as a crucial gasotransmitter involving in various physiological processes in plants. H2S signaling has been reported to involve in regulating seed germination, but the underlying mechanism remains poorly understood. Here, we found that endogenous H2S production was activated in germinating Arabidopsis seeds, correlating with upregulated both the transcription and the activity of L-cysteine desulfhydrase (EC 4.4.1.28, LCD and DES1) responsible for H2S production. Moreover, seed germination could be significantly accelerated by exogenous NaHS (the H2S donor) fumigation and over-expressing DES1, while H2S-generation defective (lcd/des1) seeds exhibited decreased germination speed. We also confirmed that the alternative oxidase (AOX), a cyanide-insensitive terminal oxidase, can be stimulated by imbibition. Furthermore, exogenous H2S fumigation and over-expressing DES1 could significantly reinforced imbibition induced increase of both the AOX1A expression and AOX protein abundance, while this increase could be obviously weakened in lcd/des1. Additionally, exogenous H2S fumigation mediated post-translational modification to keep AOX in its reduced and active state, which might involve H2S induced improvement of the reduced GSH content and the cell reducing power. The promotive effect of H2S on germination was clearly impaired by inducing aox1a mutation, indicating that AOX acts downstream of H2S signaling to accelerate seed germination. Consequently, H2S signaling was activated during germination then acted as a trigger to induce AOX mediated cyanide-resistant respiration to accelerate seed germination. Our study correlates H2S signaling to cyanide-resistant respiration, providing evidence for more extensive studies of H2S signaling.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Gasotransmissores , Sulfeto de Hidrogênio , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cianetos/metabolismo , Cianetos/farmacologia , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/farmacologia , Gasotransmissores/metabolismo , Germinação , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Proteínas Mitocondriais , Oxirredutases/metabolismo , Proteínas de Plantas , Respiração , Sementes/metabolismoRESUMO
The contamination of agricultural soils with Arsenic (As) is a significant environmental stress that restricts plant growth, metabolism, and productivity worldwide. The present study examined the role of elemental sulfur (S0) in protecting Brassica napus plants from Arsenic (As) toxicity. Arsenic (100, and 200 mg As kg-1 soil) in soil caused detrimental effects on five Brassica napus cultivars (Neelam, Teri-Uttam Jawahar, Him Sarson, GSC-101, and NUDB 26-11). The As toxicity inhibited the growth and photosynthesis indices in all cultivars with more deterioration effects in NUDB 26-11. Plant absorption and uptake of As caused the generation of oxidative injury by accumulating the reactive oxygen species (ROS), which simultaneously decreased the plant defence capability and ultimately the photosynthesis. Application of sulfur (S0, 100 or 200 mg S kg-1 soil) alleviated the negative impacts and toxicity of As on the photosynthesis and growth matrices of plants, especially under high S level. S0 also boosted the antioxidant potential of plants and toned-down lipid peroxidation and ROS aggravation such as superoxide anion (O2â¢-) and H2O2, hydrogen peroxide, in As affected plants. In general, S0 at 200 mg kg-1 soil more perceptibly increased the functionality of antioxidant enzymes, and non-enzymatic antioxidants, metal chelators and non-protein thiols. Further amendment of soil with S0 at fifteen days before seed sowing affected by As-induced toxic effects (added to soil at the time of sowing) considerably intensified the endogenous hydrogen sulfide (H2S) content and its regenerating enzymes D-cysteine desulfhydrase (DCD) and L-cysteine desulfhydrase (LCD) that further strengthened the defense capability of plants to withstand As-stress. Our results suggest the role of H2S in the S-induced defense operation of the B. napus plants in restraining As toxicity. The current study shows that S0 as a source of S might be used to promote the growth of B. napus plants in polluted agricultural soils.
Assuntos
Arsênio , Brassica napus , Antioxidantes/metabolismo , Arsênio/toxicidade , Brassica napus/metabolismo , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/farmacologia , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Fotossíntese , Espécies Reativas de Oxigênio/metabolismo , Solo , Compostos de Sulfidrila , Enxofre/metabolismoRESUMO
High glucose (HG)-induced dysfunction of vascular endothelial cells plays a crucial role in the development of diabetic vascular complications. Inhibition of cystathionine γ-synthase/hydrogen sulfide (CSE/H2 S) pathway is one of the causes of vascular endothelial cell injury induced by HG. Dopamine D1 receptors (DR1) are widely expressed and regulate important physiological functions in the vascular system. However, the effect of DR1 inhibition on HG-induced vascular endothelial apoptosis by regulating the CSE/H2 S pathway is unclear. Therefore, we aimed to determine if DR1 can regulate the CSE/H2 S pathway and regulate the effect of DR1 on HG-induced apoptosis in human umbilical vein endothelial cells. In this study, we found that HG treatment significantly decreased the expression of DR1 and CSE and the endogenous content of H2 S; DR1 agonist SKF 38393 reversed these effects, while sodium hydrosulfide (NaHS) only increased CSE expression and the endogenous H2 S production and had no effect on DR1 expression. Meanwhile, HG significantly increased the intracellular calcium concentration ([Ca2+ ]i ), and SKF 38393 further increased HG-induced [Ca2+ ]i . In addition, HG increased the lactate dehydrogenase activity, malondialdehyde and reactive oxygen species contents, apoptotic rate, the expression of cleaved caspase-3, caspase-9, and cytochrome c, and the activity of phosphorylated-inhibitor of nuclear factor-kappaBα (NF-κBα) (p-IκBα) and phosphorylated-NF-κB (p-NF-κB), and reduced cell viability, superoxide dismutase activity, and Bcl-2 expressions. SKF 38393 and NaHS markedly reversed the effect of HG. The effect of SKF 38393 was similar to N-acetyl- l-cysteine (an inhibitor of oxidative stress) or pyrrolidinedithiocarbamate ammonium (an NF-kB inhibitor). Taken together, DR1 upregulates the CSE/H2 S pathway by increasing the [Ca2+ ]i , which inhibits HG-induced apoptosis via downregulating NF-κB/IκBα pathway in vascular endothelial cells.
Assuntos
Cistationina gama-Liase , Sulfeto de Hidrogênio , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/metabolismo , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Apoptose , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/farmacologia , Dopamina/metabolismo , Glucose/metabolismo , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Sulfeto de Hidrogênio/farmacologia , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/metabolismo , Receptores de Dopamina D1/metabolismoRESUMO
BACKGROUND: Endothelial dysfunction plays a crucial role in diabetic vascular complications. A decrease in hydrogen sulfide (H2S) levels is increasingly becoming a vital factor contributing to high glucose (HG)-induced endothelial dysfunction. Dopamine D1-like receptors (DR1) activation has important physiological functions in the cardiovascular system. H2S decreases the dysfunction of vascular endothelial cells. However, no studies have reported whether DR1 protects the function of vascular endothelial cells by regulating H2S levels. AIM: The present study aimed to determine whether DR1 regulates the levels of endogenous H2S, which exerts protective effects against HG-induced injury of human umbilical vein endothelial cells (HUVECs) via Ras homolog gene family member A (RhoA)/Rho-associated coiled-coil containing kinase 1 (ROCK1) signalling. METHODS: HUVECs were exposed to HG (30 mM) or normal glucose (5.5 mM) after different treatments. Cell viability, proliferation and migration were measured by Cell Counting Kit-8, EdU cell proliferation assay, transwell assay and wound healing assay, respectively. H2S probe (7-Azido-4-Methylcoumarin) was used to detect levels of H2S. The intracellular calcium concentration ([Ca2+]i) were measured using Fluo-4 AM. The protein expressions were quantified by Western blot. RESULTS: We found that HG decreased the expression of DR1 and cystathionine γ-lyase (CSE) and H2S production. The DR1 agonist SKF38393 significantly increased DR1 and CSE expression and H2S production, whereas NaHS (a H2S donor) only increased CSE expression and H2S production but had no effect on DR1 expression. Meanwhile, SKF38393 further increased the [Ca2+]i induced by HG. In addition, HG reduced cell viability and the expression of Cyclin D1 and proliferating cell nuclear antigen and increased the expression of p21Câ¢iâ¢p/Wâ¢Aâ¢F-1, collagen I, collagen III, matrix metalloproteinase 9, osteopontin and α-smooth muscle actin and the activity of phosphorylated RhoA and ROCK1. SKF38393 and NaHS reversed these effects of HG. PPG (a CSE inhibitor) abolished the beneficial effect of SKF38393. These effects of SKF38393 were similar to those of Y-27632 (a ROCK inhibitor). CONCLUSION: Taken together, our results suggest that DR1 activation upregulates the CSE/H2S pathway by increasing the [Ca2+]i, which protects endothelial cells from HG-induced injury by inhibiting the RhoA/ROCK1 pathway.
Assuntos
Sulfeto de Hidrogênio , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Cistationina gama-Liase/genética , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/farmacologia , Glucose/toxicidade , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Quinases Associadas a rho/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/farmacologiaRESUMO
Extensive studies in prostate cancer and other malignancies have revealed that l-methionine (l-Met) and its metabolites play a critical role in tumorigenesis. Preclinical and clinical studies have demonstrated that systemic restriction of serum l-Met, either via partial dietary restriction or with bacterial l-Met-degrading enzymes exerts potent antitumor effects. However, administration of bacterial l-Met-degrading enzymes has not proven practical for human therapy because of problems with immunogenicity. As the human genome does not encode l-Met-degrading enzymes, we engineered the human cystathionine-γ-lyase (hMGL-4.0) to catalyze the selective degradation of l-Met. At therapeutically relevant dosing, hMGL-4.0 reduces serum l-Met levels to >75% for >72 h and significantly inhibits the growth of multiple prostate cancer allografts/xenografts without weight loss or toxicity. We demonstrate that in vitro, hMGL-4.0 causes tumor cell death, associated with increased reactive oxygen species, S-adenosyl-methionine depletion, global hypomethylation, induction of autophagy, and robust poly(ADP-ribose) polymerase (PARP) cleavage indicative of DNA damage and apoptosis.
Assuntos
Cistationina gama-Liase/farmacologia , Metionina/antagonistas & inibidores , Mutagênese Sítio-Dirigida , Neoplasias da Próstata/tratamento farmacológico , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Cistationina gama-Liase/genética , Cistationina gama-Liase/isolamento & purificação , Cistationina gama-Liase/uso terapêutico , Dano ao DNA/efeitos dos fármacos , Ensaios Enzimáticos , Humanos , Masculino , Metionina/sangue , Metionina/metabolismo , Camundongos , Poli(ADP-Ribose) Polimerases/metabolismo , Neoplasias da Próstata/sangue , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Testes de Toxicidade Aguda , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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 MutantesRESUMO
Hydrogen sulfide (H2S) and NO are important gasotransmitters, but how endogenous H2S affects the circulatory system has remained incompletely understood. Here, we show that CTH or CSE (cystathionine γ-lyase)-produced H2S scavenges vascular NO and controls its endogenous levels in peripheral arteries, which contribute to blood pressure regulation. Furthermore, eNOS (endothelial NO synthase) and phospho-eNOS protein levels were unaffected, but levels of nitroxyl were low in CTH-deficient arteries, demonstrating reduced direct chemical interaction between H2S and NO. Pretreatment of arterial rings from CTH-deficient mice with exogenous H2S donor rescued the endothelial vasorelaxant response and decreased tissue NO levels. Our discovery that CTH-produced H2S inhibits endogenous endothelial NO bioavailability and vascular tone is novel and fundamentally important for understanding how regulation of vascular tone is tailored for endogenous H2S to contribute to systemic blood pressure function.
Assuntos
Pressão Sanguínea/fisiologia , Cistationina gama-Liase/farmacologia , Sulfeto de Hidrogênio/metabolismo , Hipertensão/metabolismo , Óxido Nítrico/metabolismo , Vasodilatação/efeitos dos fármacos , Animais , Disponibilidade Biológica , Pressão Sanguínea/efeitos dos fármacos , Modelos Animais de Doenças , Hipertensão/fisiopatologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiopatologia , CamundongosRESUMO
Cancer cells experience higher oxidative stress from reactive oxygen species (ROS) than do non-malignant cells because of genetic alterations and abnormal growth; as a result, maintenance of the antioxidant glutathione (GSH) is essential for their survival and proliferation. Under conditions of elevated ROS, endogenous L-cysteine (L-Cys) production is insufficient for GSH synthesis. This necessitates uptake of L-Cys that is predominantly in its disulfide form, L-cystine (CSSC), via the xCT(-) transporter. We show that administration of an engineered and pharmacologically optimized human cyst(e)inase enzyme mediates sustained depletion of the extracellular L-Cys and CSSC pool in mice and non-human primates. Treatment with this enzyme selectively causes cell cycle arrest and death in cancer cells due to depletion of intracellular GSH and ensuing elevated ROS; yet this treatment results in no apparent toxicities in mice even after months of continuous treatment. Cyst(e)inase suppressed the growth of prostate carcinoma allografts, reduced tumor growth in both prostate and breast cancer xenografts and doubled the median survival time of TCL1-Tg:p53-/- mice, which develop disease resembling human chronic lymphocytic leukemia. It was observed that enzyme-mediated depletion of the serum L-Cys and CSSC pool suppresses the growth of multiple tumors, yet is very well tolerated for prolonged periods, suggesting that cyst(e)inase represents a safe and effective therapeutic modality for inactivating antioxidant cellular responses in a wide range of malignancies.
Assuntos
Neoplasias da Mama/metabolismo , Cistationina gama-Liase/farmacologia , Cisteína/efeitos dos fármacos , Cistina/efeitos dos fármacos , Leucemia Linfocítica Crônica de Células B/metabolismo , Polietilenoglicóis/farmacologia , Neoplasias da Próstata/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Cisteína/metabolismo , Cistina/metabolismo , Feminino , Glutationa/metabolismo , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Macaca fascicularis , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Transplante de Neoplasias , Estresse Oxidativo , Proteína Supressora de Tumor p53/genéticaRESUMO
OBJECTIVE: To investigate the changes in hydrogen sulfide (H2S) and cystathionine gamma-lyase (CSE) in rats with severe burn, and to analyze the effects on important organs. METHODS: One hundred and four healthy male SD rats were divided into normal control group (NC, n = 8), burn group (B, n = 48), and H2S intervention group (HI, n = 48) according to the random number table. SD rats in HI group were intraperitoneally injected with NaHS (56 micromol/kg) once a day for 5 days. Then rats in HI and B groups were subjected to 30% TBSA full-thickness burn. Blood sample as well as heart, liver, lung, kidney, and stomach tissue samples were harvested from rats in B group at post burn hour (PBH) 2, 6, 12, 24, 48, and 96 respectively for determination of serum content of H2S and CSE activity. Serum content of alanine transaminase (ALT), aspartate aminotransferase (AST), MB isoenzyme of creatine kinase (CK-MB), urea nitrogen (BUN), and creatinine (Cr) in HI and B groups were examined at each time point. Samples were harvested from above organs in each group for histomorphological observation. Above-mentioned indexes were also determined in NC group as control. Data were processed with SNK- q test, t test, correlation analysis (between serum content of H2S and CSE activities, biochemical indexes). RESULTS: Serum content of H2S and CSE activities of above organs (except for lung tissue at PBH 48, 96) in B group within PBH 96 were lower than those in NC group, reaching minimum values at PBH 6 or 12. Compared with those in NC group, serum contents of all biochemical indexes in B group were obviously increased within PBH 48, in which serum contents of BUN [(32.5 +/- 9.8) mmol/L] and Cr [(65 +/- 9) micromol/L] reached peak at PBH 6, and serum contents of ALT [(423 +/- 59) U/L], AST [(993 +/- 60) U/L], and CK-MB [(49 261 +/- 6637) U/L] peaked at PBH 12. Serum contents of all biochemical indexes in HI group at each time point were significantly decreased as compared with those in B group, but the same change tendencies were showed in both groups. Histomorphological observation showed that all the organs were severely injured in B group at PBH 24, whereas those in HI group were markedly ameliorated. Serum content of H2S in B group was respectively correlated with CSE activities of all organs (with r value from 0.639 to 0.894, P values all below 0.005) and serum contents of biochemical indexes (with r value from 0.301 to 0.585, P values all below 0.001). CONCLUSIONS: H2S/CSE system may take part in pathophysiological process in rats with severe burn. Exogenous H2S replacement therapy can protect important organs of rats with severe burn.
Assuntos
Queimaduras/patologia , Sulfeto de Hidrogênio/farmacologia , Animais , Queimaduras/tratamento farmacológico , Cistationina gama-Liase/farmacocinética , Cistationina gama-Liase/farmacologia , Sulfeto de Hidrogênio/uso terapêutico , Rim/patologia , Fígado/patologia , Pulmão/patologia , Masculino , Miocárdio/patologia , Ratos , Ratos Sprague-Dawley , Estômago/patologiaRESUMO
The combined exposure to environmental toxicants such as heavy metals and radiation is an important research area in health protection. Here we explored cadmium induced radioadaptive response (RAR) and investigated the role of hydrogen sulfide (H(2)S) and ATM kinase in this response. Our data showed that the cadmium ions with a sub-lethal concentration could induce RAR in Chang liver cells towards subsequent γ-irradiation and this response could be abrogated by DL-propargylglycine (PPG), the endogenous H(2)S synthetase inhibitor of cystathionine γ-lyase (CSE), but not by aminooxyacetic acid (AOAA), the inhibitor of cystathionine ß-synthase (CBS). Moreover, the pretreatment of cells with NaHS also stimulated cellular adaptive response to radiation. Both cadmium treatment and irradiation up-regulated the expression of CSE protein in a time-dependent manner but had no influence on the expression of CBS protein. In the primed cells, the time course of CBS expression showed no significant difference with the cells treated with 2Gy irradiation alone, however, the CSE expression was easier to reach the maximum level, indicating a more efficient H(2)S production by CSE. Moreover, the cadmium-induced RAR was totally suppressed by KU-55933, a specific ATM inhibitor that did not change the CSE expression after radiation. However, exogenous H(2)S decreased the phosphorylation level of radiation-induced ATM. In conclusion, the present results demonstrate firstly that H(2)S is involved in the cadmium induced cross-adaptive response to challenging radiation. CSE, rather than CBS, may mainly responsible for the H(2)S production during this RAR which may also be mediated by ATM pathway. However, the activation of CSE is independent of ATM but could negatively regulate the phosphorylation of ATM.
Assuntos
Adaptação Fisiológica/efeitos da radiação , Cádmio/farmacologia , Proteínas de Ciclo Celular/farmacologia , Cistationina gama-Liase/farmacologia , Proteínas de Ligação a DNA/farmacologia , Sulfeto de Hidrogênio/metabolismo , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas Supressoras de Tumor/farmacologia , Adaptação Fisiológica/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Linhagem Celular , Cistationina gama-Liase/metabolismo , Humanos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiaçãoRESUMO
Hydrogen sulfide (H2S) has been shown recently to function as an important gasotransmitter. The present study investigated the vascular effects of H2S, both exogenously applied and endogenously generated, on resistance mesenteric arteries of rats and the underlying mechanisms. Both H2S and NaHS evoked concentration-dependent relaxation of in vitro perfused rat mesenteric artery beds (MAB). The sensitivity of MAB to H2S (EC50, 25.2 +/- 3.6 microM) was about fivefold higher than that of rat aortic tissues. Removal of endothelium or coapplication of charybdotoxin and apamin to endothelium-intact MAB significantly reduced the vasorelaxation effects of H2S. The H2S-induced relaxation of MAB was partially mediated by ATP-sensitive K+ (KATP) channel activity in vascular smooth muscle cells. Pinacidil (EC50, 1.7 +/- 0.1 microM, n=6) mimicked, but glibenclamide (10 microM, n=6) suppressed, the vasorelaxant effect of H2S. KATP channel currents in isolated mesenteric artery smooth muscle cells were significantly augmented by H2S. L-cysteine, a substrate of cystathionine-gamma-lyase (CSE), at 1 mM increased endogenous H2S production by sixfold in rat mesenteric artery tissues and decreased contractility of MAB. DL-propargylglycine (a blocker of CSE) at 10 microM abolished L-cysteine-dependent increase in H2S production and relaxation of MAB. Our results demonstrated a tissue-specific relaxant response of resistance arteries to H2S. The stimulation of KATP channels in vascular smooth muscle cells and charybdotoxin/apamin-sensitive K+ channels in vascular endothelium by H2S represents important cellular mechanisms for H2S effect on MAB. Our study also demonstrated that endogenous CSE can generate sufficient H2S from exogenous L-cysteine to cause vasodilation. Future studies are merited to investigate direct contribution of endogenous H2S to regulation of vascular tone.
Assuntos
Sulfeto de Hidrogênio/farmacologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/fisiologia , Vasodilatação , Vasodilatadores/farmacologia , Trifosfato de Adenosina/metabolismo , Animais , Cistationina gama-Liase/farmacologia , Sulfeto de Hidrogênio/metabolismo , Técnicas In Vitro , Masculino , Artérias Mesentéricas/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Fenilefrina/farmacologia , Canais de Potássio/metabolismo , Canais de Potássio/fisiologia , Ratos , Ratos Sprague-Dawley , Sulfetos/farmacologia , Resistência Vascular , Vasoconstritores/farmacologia , Vasodilatação/fisiologia , Vasodilatadores/metabolismoRESUMO
Liver cytosolic gamma-cystathionase catalyzes the generation of reduced sulfur species, referred to as "bound sulfur,' in the presence of cystine. Incubating a rat liver cytosol fraction in the presence of cystine or oxidized glutathione inactivated certain cytosolic enzyme activities. The activities of cytosolic phosphofructokinase (PFK) and pyruvate kinase rapidly decreased at pH 7.4 during incubation with a lower concentration of cystine than during incubation with oxidized glutathione. Hexokinase and 11 other enzymes in the system were affected minimally or not at all. Adding dithiothreitol to the system reactivated the modified enzymes. Inactivated PFK activity could also be recovered when reduced glutathione or NADPH was added to the cytosol fraction. In these reconstitution systems, purified rat liver PFK was directly inactivated with cystine trisulfide (one of the low molecular types of bound sulfur), but not by cystine (below 0.1 mM). Purified PFK was also inactivated by incubation with cystine plus gamma-cystathionase freshly prepared from cytosol. This was not observed, however, when gamma-cystathionase was pretreated with a specific inhibitor, D,L-propargylglycine. The cystine-dependent inactivation of PFK observed in liver cytosol is shown to be caused mainly by the reaction between bound sulfur and the enzyme, but not by the direct thiol/disulfide exchange. Thus, in vitro modification of the cytosolic enzymes by bound sulfur generated from cystine with gamma-cystathionase has high potency and relatively specific.
Assuntos
Cistationina gama-Liase/farmacologia , Cistina/farmacologia , Fígado/enzimologia , Animais , Citosol/enzimologia , Ditiotreitol , Gluconeogênese , Glutationa , Glicólise , Masculino , Fosfofrutoquinase-1/antagonistas & inibidores , Piruvato Quinase/antagonistas & inibidores , Ratos , Ratos Wistar , Enxofre/químicaRESUMO
The results of several in vitro studies have suggested that the enzyme cystathionase gamma-lyase (EC 4.4.1.1) may function in the endogenous detoxification of cyanide; however, this possibility has not been investigated in vivo. If cystathionase gamma-lyase in involved in the endogenous detoxification of cyanide, it logically follows that inhibiting cystathionase gamma-lyase should increase the toxicity of cyanide. To test this hypothesis, the activity of cystathionase gamma-lyase was inhibited with a suicide inhibitor, 2-amino-4-pentynoic acid (propargyl-glycine). The activity of liver cystathionase gamma-lyase activity was decreased 96.8% by administration of propargylglycine, indicating that the propargylglycine treatment was effective. The propargylglycine treatment did not alter the activity of thiosulfate:cyanide sulfurtransferase (EC 2.8.1.1) or 3-mercaptopyruvate:cyanide sulfurtransferase (EC 2.8.1.2), two other enzymes that have been proposed to be involved in the detoxification of cyanide. The LD50 of cyanide in rats treated with propargylglycine was 5.14 +/- 0.029 mg NaCN/kg, which was significantly (P < 0.05) lower than the 5.98 +/- 0.008 mg NaCN/kg LD50 of cyanide determined in control rats. The results of these studies suggest that cystathionase gamma-lyase may participate in the detoxification of cyanide in vivo.
Assuntos
Cianetos/metabolismo , Cistationina gama-Liase/farmacologia , Fígado/efeitos dos fármacos , Liases/farmacologia , Animais , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Tyrosine aminotransferase is stable in homogenates of rat liver, but not when L-cystine or L-cysteine is added, which causes the enzyme to be reversibly inactivated due to oxidation of thiol groups. By monitoring inactivation of the aminotransferase in the presence of L-cystine, a factor responsible for this loss of activity was purified from rat liver. The factor required vitamin B6 and co-purified with gamma-cystathionase during numerous steps. Highly purified inactivating factor contained a protein that was identical in size and isoelectric point to cystathionase but also contained a dissimilar peptide that appeared to be unrelated to cystathionase. Cystathionase and the cystine-dependent inactivator shared several catalytic activities, including the hydrolysis of cystathionine, desulfuration of cystine, and desulfhydration of cysteine. During incubation of L-cysteine with the purified factor, hydrogen sulfide was generated but no inactivation of the aminotransferase occurred, suggesting that cysteine-dependent inactivation requires additional mechanisms. An insoluble inactivator of tyrosine aminotransferase that is produced during the reaction may be elemental sulfur, since colloidal suspensions of sulfur also inhibited the enzyme. Another inhibitor fractionated with high molecular weight substances; this may be protein-bound sulfane.
Assuntos
Cistationina gama-Liase/isolamento & purificação , Cistina/farmacologia , Liases/isolamento & purificação , Tirosina Transaminase/antagonistas & inibidores , Animais , Cromatografia , Cistationina gama-Liase/metabolismo , Cistationina gama-Liase/farmacologia , Cisteína/metabolismo , Cisteína/farmacologia , Cistina/metabolismo , Feminino , Focalização Isoelétrica , Ponto Isoelétrico , Cinética , Fígado/análise , Masculino , Fosfato de Piridoxal/farmacologia , Piridoxina/farmacologia , Ratos , Ratos EndogâmicosRESUMO
The L-cyst(e)ine requirements of normal and malignant cells are reviewed and expanded within the context of establishing whether the measurement of gamma-cystathionase levels constitutes a predictive test for tumor sensitivity to L-cyst(e)ine depletion. The ability of both purified L-cysteine desulfhydrase and gamma-cystathionase to inhibit the growth of the L-cystine-dependent L1210 leukemia in culture is presented, as well as approaches to circumvent the limitations of these enzymes for in vivo therapy. The ability of proparagylglycine to inhibit L-cysteine biosynthesis in vivo is reviewed for its possible use in combination therapy. In addition, the ability of poly D,L-alanine modification of Escherichia coli L-asparaginase to increase the plasma half-life in mice tenfold as well as to decrease the immunogenicity of the enzyme is presented.