RESUMEN

Previously, the direct interactions of Bß26-42 fibrin residues with prothrombin were demonstrated. It was also shown that forming prothrombin complexes with E- or DDE-fragments causes non-enzymatic prothrombin activation. The direct measuring of the prothrombin level in the blood plasma of patients with acute myocardial infarction (AMI) allowed us to find a situation where such an activation can occur in vivo. Blood coagulation parameters in the blood plasma of patients with AMI were measured at 2 hours, three days, and seven days after the thrombolysis by streptokinase accompanied with intravenous administration of anticoagulants: unfractionated high molecular weight heparin (HMWH) and low-molecular-weight heparin (LMWH). The prothrombin level in the blood plasma of patients with AMI was normal before thrombolytic therapy and substantially decreased after streptokinase administration. This effect was prominent in the case of concomitant anticoagulant therapy with LMWH and was not observed when HMWH was applied. It can be explained by the fact that LMWH preferentially inhibits factor Xa, while the HMWH is an effective inhibitor of both factor Xa and thrombin. This observation suggested that the prothrombin level decrease was caused by the thrombin-like activity and possible autolysis of prothrombin by thrombin. Also, thrombolytic therapy with streptokinase caused the accumulation of fibrin degradation products (FDPs), some of which were able to bind prothrombin. The dramatic decrease of prothrombin level in the blood plasma of patients with AMI during thrombolysis allowed us to conclude the non-enzymatic prothrombin activation with the following autolysis of prothrombin that contributes to the pathology.

Asunto(s)

Infarto del Miocardio , Protrombina , Humanos , Protrombina/metabolismo , Protrombina/uso terapéutico , Heparina de Bajo-Peso-Molecular/uso terapéutico , Trombina , Factor Xa/uso terapéutico , Infarto del Miocardio/tratamiento farmacológico , Heparina/farmacología , Heparina/uso terapéutico , Estreptoquinasa/uso terapéutico , Estreptoquinasa/farmacología , Terapia Trombolítica , Anticoagulantes/uso terapéutico

RESUMEN

Soluble fibrin is composed mainly of desA fibrin and fibrinogen oligomers consisting of fewer than 16 monomers partially cross-linked by factor XIIIa. Soluble fibrin cannot stimulate Glu-plasminogen activation by tissue plasminogen activator (t-PA); therefore, it may not be a direct predecessor of D-dimer. However, within the microcirculatory system, soluble fibrin oligomers may form microclots. Fibrin microclots stimulate Glu-plasminogen activation by t-PA, a process resulting in the formation of Glu-plasmin. Glu-plasmin dissolves the microclots, forming D-dimer. In normal and pathological blood plasma samples, soluble fibrin levels are substantially higher than those of D-dimer. Their concentrations in the plasma are also regulated by transendothelial transfer, absorption by blood macrophages, and binding and internalization with low-density lipoprotein receptors of the cells of the reticuloendothelial system. Therefore, the exact mechanisms of fibrin clots formation and elimination in normal and pathological conditions remain unclear. In this study, we reviewed findings on the molecular mechanisms of the formation and dissolution of fibrin clots, fibrin-dependent activation of Glu-plasminogen by t-PA, and blood plasma behavior in the microcirculatory system. Finally, we proposed a model that explains the relations of D-dimer and soluble fibrin underlying the common and separate mechanisms of their formation and elimination.

Asunto(s)

Fibrinolisina , Activador de Tejido Plasminógeno , Humanos , Activador de Tejido Plasminógeno/metabolismo , Fibrinolisina/metabolismo , Microcirculación , Plasminógeno/metabolismo , Fibrina/metabolismo

RESUMEN

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.

Asunto(s)

Ácido Aminooxiacético/farmacología , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Cistationina betasintasa/antagonistas & inhibidores , Profármacos/farmacología , Animales , Células HCT116 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos

RESUMEN

OBJECTIVE: Activation of the constitutive nuclear and mitochondrial enzyme poly (ADP-ribose) polymerase (PARP) has been implicated in the pathogenesis of cell dysfunction, inflammation, and organ failure in various forms of critical illness. The objective of our study was to evaluate the efficacy and safety of the clinically approved PARP inhibitor olaparib in an experimental model of pancreatitis in vivo and in a pancreatic cell line subjected to oxidative stress in vitro. The preclinical studies were complemented with analysis of clinical samples to detect PARP activation in pancreatitis. METHODS: Mice were subjected to cerulein-induced pancreatitis; circulating mediators and circulating organ injury markers; pancreatic myeloperoxidase and malondialdehyde levels were measured and histology of the pancreas was assessed. In human pancreatic duct epithelial cells (HPDE) subjected to oxidative stress, PARP activation was measured by PAR Western blotting and cell viability and DNA integrity were quantified. In clinical samples, PARP activation was assessed by PAR (the enzymatic product of PARP) immunohistochemistry. RESULTS: In male mice subjected to pancreatitis, olaparib (3 mg/kg i.p.) improved pancreatic function: it reduced pancreatic myeloperoxidase and malondialdehyde levels, attenuated the plasma amylase levels, and improved the histological picture of the pancreas. It also attenuated the plasma levels of pro-inflammatory mediators (TNF-α, IL-1ß, IL-2, IL-4, IL-6, IL-12, IP-10, KC) but not MCP-1, RANTES, or the anti-inflammatory cytokine IL-10. Finally, it prevented the slight, but significant increase in plasma blood urea nitrogen level, suggesting improved renal function. The protective effect of olaparib was also confirmed in female mice. In HPDE cells subjected to oxidative stress olaparib (1 µM) inhibited PARP activity, protected against the loss of cell viability, and prevented the loss of cellular NAD levels. Olaparib, at 1µM to 30 µM did not have any adverse effects on DNA integrity. In human pancreatic samples from patients who died of pancreatitis, increased accumulation of PAR was demonstrated. CONCLUSION: Olaparib improves organ function and tempers the hyperinflammatory response in pancreatitis. It also protects against pancreatic cell injury in vitro without adversely affecting DNA integrity. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of pancreatitis.

Asunto(s)

Pancreatitis/tratamiento farmacológico , Pancreatitis/patología , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Animales , Técnicas de Cultivo de Célula , Línea Celular , Ceruletida , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Femenino , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Conductos Pancreáticos/efectos de los fármacos , Conductos Pancreáticos/patología , Pancreatitis/etiología

RESUMEN

Poly(ADP-ribose) polymerase (PARP) is involved in the pathogenesis of cell dysfunction, inflammation and organ failure during septic shock. The goal of the current study was to investigate the efficacy and safety of the clinically approved PARP inhibitor olaparib in experimental models of oxidative stress in vitro and in sepsis in vivo. In mice subjected to cecal ligation and puncture (CLP) organ injury markers, circulating and splenic immune cell distributions, circulating mediators, DNA integrity and survival was measured. In U937 cells subjected to oxidative stress, cellular bioenergetics, viability and DNA integrity were measured. Olaparib was used to inhibit PARP. The results show that in adult male mice subjected to CLP, olaparib (1-10 mg/kg i.p.) improved multiorgan dysfunction. Olaparib treatment reduced the degree of bacterial CFUs. Olaparib attenuated the increases in the levels of several circulating mediators in the plasma. In the spleen, the number of CD4+ and CD8+ lymphocytes were reduced in response to CLP; this reduction was inhibited by olaparib treatment. Treg but not Th17 lymphocytes increased in response to CLP; these cell populations were reduced in sepsis when the animals received olaparib. The Th17/Treg ratio was lower in CLP-olaparib group than in the CLP control group. Analysis of miRNA expression identified a multitude of changes in spleen and circulating white blood cell miRNA levels after CLP; olaparib treatment selectively modulated these responses. Olaparib extended the survival rate of mice subjected to CLP. In contrast to males, in female mice olaparib did not have significant protective effects in CLP. In aged mice olaparib exerted beneficial effects that were less pronounced than the effects obtained in young adult males. In in vitro experiments in U937 cells subjected to oxidative stress, olaparib (1-100 µM) inhibited PARP activity, protected against the loss of cell viability, preserved NAD+ levels and improved cellular bioenergetics. In none of the in vivo or in vitro experiments did we observe any adverse effects of olaparib on nuclear or mitochondrial DNA integrity. In conclusion, olaparib improves organ function and extends survival in septic shock. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of septic shock.

Asunto(s)

Antiinflamatorios/uso terapéutico , Ftalazinas/uso terapéutico , Piperazinas/uso terapéutico , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Sepsis/tratamiento farmacológico , Animales , Antiinflamatorios/farmacología , Ciego , Citocinas/sangre , ADN/efectos de los fármacos , Reposicionamiento de Medicamentos , Femenino , Humanos , Ligadura , Hígado/efectos de los fármacos , Hígado/patología , Pulmón/efectos de los fármacos , Pulmón/patología , Recuento de Linfocitos , Masculino , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Ftalazinas/farmacología , Piperazinas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Punciones , Sepsis/sangre , Sepsis/inmunología , Sepsis/patología , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Células U937

RESUMEN

The gaseous transmitter hydrogen sulfide (H2S) has been implicated in various forms of critical illness. Here, we have compared the outcome of scald burn injury in wild-type mice and in mice deficient in 3-mercaptopyruvate sulfurtransferase (3-MST), a mammalian H2S-generating enzyme. Outcome variables included indices of organ injury, clinical chemistry parameters, and plasma levels of inflammatory mediators. Plasma levels of H2S significantly increased in response to burn in wild-type mice, but remained unchanged in 3-MST-/- mice. The capacity of tissue homogenates to produce H2S from 3-mercaptopyruvate was unaffected by burn injury. In 3-MST-/- mice, compared to wild-type controls, there was a significant enhancement in the accumulation of polymorphonuclear cells (as assessed by the quantification of myeloperoxidase) in the liver (but not heart, lung, or skin) at 7 days postburn. Oxidative tissue damage (as assessed by malon dialdehyde content) was comparable between wild-type and 3-MST-deficient mice in all tissues studied. 3-MST-/- and wild-type mice exhibited comparable burn-induced elevations in circulating plasma levels of hepatic injury; however, 3-MST-/- mice exhibited a higher degree of renal injury (as reflected by elevated blood urea nitrogen levels) at 7 days postburn. Inflammatory mediators (eg, TNF-α, IL-1ß, IL-2, IL-6, IL-10, and IL-12) increased in burn injury, but without significant differences between the 3-MST-/- and wild-type groups. The healing of the burn wound was also unaffected by 3-MST deficiency. In conclusion, the absence of the H2S-producing enzyme 3-MST slightly exacerbates the development of multiorgan dysfunction but does not affect inflammatory mediator production or wound healing in a murine model of burn injury.

Asunto(s)

Quemaduras/enzimología , Inflamación/enzimología , Insuficiencia Multiorgánica/enzimología , Sulfurtransferasas/deficiencia , Cicatrización de Heridas , Animales , Modelos Animales de Enfermedad , Mediadores de Inflamación/sangre , Masculino , Ratones

RESUMEN

Hydrogen sulfide (H2S) production in colon cancer cells supports cellular bioenergetics and proliferation. The aim of the present study was to investigate the alterations in H2S homeostasis during the development of resistance to 5-fluorouracil (5-FU), a commonly used chemotherapeutic agent. A 5-FU-resistant HCT116 human colon cancer cell line was established by serial passage in the presence of increasing 5-FU concentrations. The 5-FU-resistant cells also demonstrated a partial resistance to an unrelated chemotherapeutic agent, oxaliplatin. Compared to parental cells, the 5-FU-resistant cells rely more on oxidative phosphorylation than glycolysis for bioenergetic function. There was a significant increase in the expression of the drug-metabolizing cytochrome P450 enzymes CYP1A2 and CYP2A6 in 5-FU-resistant cells. The CYP450 inhibitor phenylpyrrole enhanced 5-FU-induced cytotoxicity in 5-FU-resistant cells. Two major H2S-generating enzymes, cystathionine-ß-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (3-MST) were upregulated in the 5-FU-resistant cells. 5-FU-resistant cells exhibited decreased sensitivity to the CBS inhibitor aminooxyacetate (AOAA) in terms of suppression of cell viability, inhibition of cell proliferation and inhibition of oxidative phosphorylation. However, 5FU-resistant cells remained sensitive to the antiproliferative effect of benserazide (a recently identified, potentially repurposable CBS inhibitor). Taken together, the current data suggest that 5-FU resistance in HCT116 cells is associated with the upregulation of drug-metabolizing enzymes and an enhancement of endogenous H2S production. The anticancer effect of prototypical H2S biosynthesis inhibitor AOAA is impaired in 5-FU-resistant cells, but benserazide remains efficacious. Pharmacological approaches aimed at restoring the sensitivity of 5-FU-resistant cells to chemotherapeutic agents may be useful in the formulation of novel therapeutic strategies against colorectal cancer.

Asunto(s)

Antineoplásicos/farmacología , Neoplasias del Colon/enzimología , Sulfuro de Hidrógeno/metabolismo , Regulación hacia Arriba , Ácido Aminooxiacético/farmacología , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/metabolismo , Cistationina betasintasa/genética , Cistationina betasintasa/metabolismo , Resistencia a Antineoplásicos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Humanos , Fosforilación , Sulfurtransferasas/genética , Sulfurtransferasas/metabolismo

RESUMEN

The trans-sulfuration enzyme cystathionine-ß-synthase (CBS) and its product hydrogen sulfide (H2S) are aberrantly upregulated in colorectal cancers, where they contribute to tumor growth and progression by both autocrine and paracrine mechanisms. However, it is unknown whether the CBS/H2S axis plays a role in colorectal carcinogenesis. Here, we report upregulation of CBS in human biopsies of precancerous adenomatous polyps and show that forced upregulation of CBS in an adenoma-like colonic epithelial cell line is sufficient to induce metabolic and gene expression profiles characteristic of colorectal cancer cells. Differentially expressed metabolites (65 increased and 20 decreased) clustered into the glycolytic pathway, nucleotide sugars, intermediates of the pentose phosphate pathway, and lipogenesis, including primarily phospholipids, sphingolipids, and bile acids. CBS upregulation induced broad changes in the NCM356 cell transcriptome with over 350 differentially expressed genes. These genes overlapped significantly with gene sets related to glycolysis, hypoxia, and a colon cancer cell phenotype, including genes regulated by NF-κB, KRAS, p53, and Wnt signaling, genes downregulated after E-cadherin knockdown, and genes related to increased extracellular matrix, cell adhesion, and epithelial-to-mesenchymal transition. The CBS-induced switch to an anabolic metabolism was associated with increased NCM356 cell bioenergetics, proliferation, invasion through Matrigel, resistance to anoikis, and CBS-dependent tumorigenesis in immunocompromised mice. Genetic ablation of CBS in CBS heterozygous mice (CBS+/- ) reduced the number of mutagen-induced aberrant colonic crypt foci. Taken together, these results establish that activation of the CBS/H2S axis promotes colon carcinogenesis. Cancer Res; 77(21); 5741-54. ©2017 AACR.

Asunto(s)

Pólipos Adenomatosos/genética , Colon/metabolismo , Cistationina betasintasa/genética , Mucosa Intestinal/metabolismo , Regulación hacia Arriba , Pólipos Adenomatosos/metabolismo , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Línea Celular , Movimiento Celular/genética , Colon/patología , Cistationina betasintasa/metabolismo , Femenino , Perfilación de la Expresión Génica/métodos , Regulación Neoplásica de la Expresión Génica , Células HCT116 , Humanos , Sulfuro de Hidrógeno/metabolismo , Mucosa Intestinal/patología , Masculino , Metabolómica/métodos , Ratones Noqueados , Ratones Desnudos , Trasplante Heterólogo

RESUMEN

Considering the role of H2S in critical illness, the aim of this study was to compare the outcome of burn in wild-type mice and in mice deficient in CSE, one of the principal mammalian H2S-generating enzymes. Animals were subjected to scald burn. Outcome variables included indices of organ injury, clinical chemistry parameters and plasma levels of inflammatory mediators. Plasma levels of H2S significantly increased in response to burn in wild-type mice, but remained unchanged in CSE-/- mice. Expression of the three H2S-producing enzymes (CSE, CBS and 3-MST) in the lung and liver, and the capacity of tissue homogenates to produce H2S, however, was not affected by burn. In CSE deficient mice there was a significant amelioration of burn-induced accumulation of myeloperoxidase levels in heart, lung, liver and kidney and significantly lower degree of malon dialdehyde accumulation in the heart, lung and kidney than in wild-type mice. CSE deficient mice, compared to wild-type mice, showed a significant attenuation of the burn-induced elevation in circulating alkaline aminotransferase and blood urea nitrogen and creatinine levels, indicative of protective effects of CSE deficiency against burn-induced hepatic, and renal functional impairment. Multiple burn-induced inflammatory mediators (TNF-α, IL-1ß, IL-4, IL-6, IL-10 and IL-12) were significantly lower in the plasma of CSE-/- animals after burn than in the plasma of wild-type controls subjected to burns. In conclusion, CSE deficiency improves organ function and attenuates the inflammatory response in a murine model of burn.

Asunto(s)

Quemaduras/complicaciones , Cistationina gamma-Liasa/deficiencia , Inflamación/metabolismo , Insuficiencia Multiorgánica/metabolismo , Análisis de Varianza , Animales , Biomarcadores/metabolismo , Western Blotting , Quemaduras/metabolismo , Cistationina gamma-Liasa/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Sulfuro de Hidrógeno/metabolismo , Masculino , Ratones

RESUMEN

Therapeutic manipulation of the gasotransmitter hydrogen sulfide (H2S) has recently been proposed as a novel targeted anticancer approach. Here we show that human lung adenocarcinoma tissue expresses high levels of hydrogen sulfide (H2S) producing enzymes, namely, cystathionine beta-synthase (CBS), cystathionine gamma lyase (CSE) and 3-mercaptopyruvate sulfurtransferase (3-MST), in comparison to adjacent lung tissue. In cultured lung adenocarcinoma but not in normal lung epithelial cells elevated H2S stimulates mitochondrial DNA repair through sulfhydration of EXOG, which, in turn, promotes mitochondrial DNA repair complex assembly, thereby enhancing mitochondrial DNA repair capacity. In addition, inhibition of H2S-producing enzymes suppresses critical bioenergetics parameters in lung adenocarcinoma cells. Together, inhibition of H2S-producing enzymes sensitize lung adenocarcinoma cells to chemotherapeutic agents via induction of mitochondrial dysfunction as shown in in vitro and in vivo models, suggesting a novel mechanism to overcome tumor chemoresistance.

Asunto(s)

Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/metabolismo , Antineoplásicos/uso terapéutico , Reparación del ADN , Metabolismo Energético , Sulfuro de Hidrógeno/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Mitocondrias/metabolismo , Adenocarcinoma/patología , Adenocarcinoma del Pulmón , Animales , Antineoplásicos/farmacología , Reparación del ADN/efectos de los fármacos , ADN Mitocondrial/genética , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Neoplasias Pulmonares/patología , Mitocondrias/efectos de los fármacos , Modelos Biológicos , Células Tumorales Cultivadas

RESUMEN

Cystathionine-ß-synthase (CBS) has been recently identified as a drug target for several forms of cancer. Currently no potent and selective CBS inhibitors are available. Using a composite collection of 8871 clinically used drugs and well-annotated pharmacological compounds (including the LOPAC library, the FDA Approved Drug Library, the NIH Clinical Collection, the New Prestwick Chemical Library, the US Drug Collection, the International Drug Collection, the 'Killer Plates' collection and a small custom collection of PLP-dependent enzyme inhibitors), we conducted an in vitro screen in order to identify inhibitors for CBS using a primary 7-azido-4-methylcoumarin (AzMc) screen to detect CBS-derived hydrogen sulfide (H2S) production. Initial hits were subjected to counterscreens using the methylene blue assay (a secondary assay to measure H2S production) and were assessed for their ability to quench the H2S signal produced by the H2S donor compound GYY4137. Four compounds, hexachlorophene, tannic acid, aurintricarboxylic acid and benserazide showed concentration-dependent CBS inhibitory actions without scavenging H2S released from GYY4137, identifying them as direct CBS inhibitors. Hexachlorophene (IC50: ∼60µM), tannic acid (IC50: ∼40µM) and benserazide (IC50: ∼30µM) were less potent CBS inhibitors than the two reference compounds AOAA (IC50: ∼3µM) and NSC67078 (IC50: ∼1µM), while aurintricarboxylic acid (IC50: ∼3µM) was equipotent with AOAA. The second reference compound NSC67078 not only inhibited the CBS-induced AzMC fluorescence signal (IC50: ∼1µM), but also inhibited with the GYY4137-induced AzMC fluorescence signal with (IC50 of ∼6µM) indicative of scavenging/non-specific effects. Hexachlorophene (IC50: ∼6µM), tannic acid (IC50: ∼20µM), benserazide (IC50: ∼20µM), and NSC67078 (IC50: ∼0.3µM) inhibited HCT116 colon cancer cells proliferation with greater potency than AOAA (IC50: ∼300µM). In contrast, although a CBS inhibitor in the cell-free assay, aurintricarboxylic acid failed to inhibit HCT116 proliferation at lower concentrations, and stimulated cell proliferation at 300µM. Copper-containing compounds present in the libraries, were also found to be potent inhibitors of recombinant CBS; however this activity was due to the CBS inhibitory effect of copper ions themselves. However, copper ions, up to 300µM, did not inhibit HCT116 cell proliferation. Benserazide was only a weak inhibitor of the activity of the other H2S-generating enzymes CSE and 3-MST activity (16% and 35% inhibition at 100µM, respectively) in vitro. Benserazide suppressed HCT116 mitochondrial function and inhibited proliferation of the high CBS-expressing colon cancer cell line HT29, but not the low CBS-expressing line, LoVo. The major benserazide metabolite 2,3,4-trihydroxybenzylhydrazine also inhibited CBS activity and suppressed HCT116 cell proliferation in vitro. In an in vivo study of nude mice bearing human colon cancer cell xenografts, benserazide (50mg/kg/days.q.) prevented tumor growth. In silico docking simulations showed that benserazide binds in the active site of the enzyme and reacts with the PLP cofactor by forming reversible but kinetically stable Schiff base-like adducts with the formyl moiety of pyridoxal. We conclude that benserazide inhibits CBS activity and suppresses colon cancer cell proliferation and bioenergetics in vitro, and tumor growth in vivo. Further pharmacokinetic, pharmacodynamic and preclinical animal studies are necessary to evaluate the potential of repurposing benserazide for the treatment of colorectal cancers.

Asunto(s)

Benserazida/farmacología , Neoplasias del Colon/tratamiento farmacológico , Cistationina betasintasa/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cumarinas/farmacología , Reposicionamiento de Medicamentos/métodos , Metabolismo Energético/efectos de los fármacos , Femenino , Células HCT116 , Células HT29 , Humanos , Hidrazinas/farmacología , Sulfuro de Hidrógeno/metabolismo , Masculino , Ratones , Ratones Desnudos , Mitocondrias/efectos de los fármacos , Morfolinas/farmacología , Compuestos Organotiofosforados/farmacología , Terapias en Investigación/métodos

RESUMEN

Colon cancer cells contain high levels of cystathionine-beta-synthase (CBS). Its product, hydrogen sulfide (H2S) promotes the growth and proliferation of colorectal tumor cells. In order to improve the antitumor efficacy of the prototypical CBS inhibitor aminooxyacetic acid (AOAA), we have designed and synthesized YD0171, a methyl ester derivative of AOAA. The antiproliferative effect of YD0171 exceeded the antiproliferative potency of AOAA in HCT116 human colon cancer cells. The esterase inhibitor paraoxon prevented the cellular inhibition of CBS activity by YD0171. YD0171 suppressed mitochondrial respiration and glycolytic function and induced G0/G1 arrest, but did not induce tumor cell apoptosis or necrosis. Metabolomic analysis in HCT116 cells showed that YD0171 affects multiple pathways of cell metabolism. The efficacy of YD0171 as an inhibitor of tumor growth was also tested in nude mice bearing subcutaneous HCT116 cancer cell xenografts. Animals were treated via subcutaneous injection of vehicle, AOAA (1, 3 or 9 mg/kg/day) or YD0171 (0.1, 0.5 or 1 mg/kg/day) for 3 weeks. Tumor growth was significantly reduced by 9 mg/kg/day AOAA, but not at the lower doses. YD0171 was more potent: tumor volume was significantly inhibited at 0.5 and 1 mg/kg/day. Thus, the in vivo efficacy of YD0171 is 9-times higher than that of AOAA. YD0171 (1 mg/kg/day) attenuated tumor growth and metastasis formation in the intracecal HCT116 tumor model. YD0171 (3 mg/kg/day) also reduced tumor growth in patient-derived tumor xenograft (PDTX) bearing athymic mice. YD0171 (3 mg/kg/day) induced the regression of established HCT116 tumors in vivo. A 5-day safety study in mice demonstrated that YD0171 at 20 mg/kg/day (given in two divided doses) does not increase plasma markers of organ injury, nor does it induce histological alterations in the liver or kidney. YD0171 caused a slight elevation in plasma homocysteine levels. In conclusion, the prodrug approach improves the pharmacological profile of AOAA; YD0171 represents a prototype for CBS inhibitory anticancer prodrugs. By targeting colorectal cancer bioenergetics, an emerging important hallmark of cancer, the approach exemplified herein may offer direct translational opportunities.

RESUMEN

Cecal ligation and puncture (CLP)-induced sepsis is a serious medical condition, caused by a severe systemic infection resulting in a systemic inflammatory response. Recent studies have suggested the therapeutic potential of donors of hydrogen sulfide (H2S), a novel endogenous gasotransmitter and biological mediator in various diseases. The aim of the present study was to assess the effect of H2S supplementation in sepsis, with special reference to its effect on the modulation of regional blood flow. We infused sodium hydrosulfide (NaHS), a compound that produces H2S in aqueous solution (1, 3, or 10 mg/kg/h, for 1 h at each dose level) in control rats or rats 24 h after CLP, and measured blood flow using fluorescent microspheres. In normal control animals, NaHS induced a characteristic redistribution of blood flow, and reduced cardiac, hepatic, and renal blood flow in a dose-dependent fashion. In contrast, in rats subjected to CLP, cardiac, hepatic, and renal blood flow was significantly reduced; infusion of NaHS (1 mg/kg/h and 3 mg/kg/h) significantly increased organ blood flow. In other words, the effect of H2S on regional blood flow is dependent on the status of the animals (i.e., a decrease in blood flow in normal controls, but an increase in blood flow in CLP). We have also evaluated the effect of delayed treatment with NaHS on organ dysfunction and the inflammatory response by treating the animals with NaHS (3 mg/kg) intraperitoneally (i.p.) at 24 h after the start of the CLP procedure; plasma levels of various cytokines and tissue indicators of inflammatory cell infiltration and oxidative stress were measured 6 h later. After 24 h of CLP, glomerular function was significantly impaired, as evidenced by markedly increased (over 4-fold over baseline) blood urea nitrogen and creatinine levels; this increase was also significantly reduced by treatment with NaHS. NaHS also attenuated the CLP-induced increases in malondialdehyde levels (an index of oxidative stress) in heart as well as in liver and myeloperoxidase levels (an index of neutrophil infiltration) in heart and lung. Plasma levels of IL-1ß, IL-5, IL-6, TNF-α, and HMGB1 were attenuated by NaHS. Treatment of NaHS at 3 mg/kg i.p. (but not 1 mg/kg or 6 mg/kg), starting 24 h post-CLP, with dosing repeated every 6 h, improved the survival rate in CLP animals. In summary, treatment with 3 mg/kg H2S-when started in a delayed manner, when CLP-induced organ injury, inflammation and blood flow redistribution have already ensued-improves blood flow to several organs, protects against multiple organ failure, and reduces the plasma levels of multiple pro-inflammatory mediators. These findings support the view that H2S donation may have therapeutic potential in sepsis.

Asunto(s)

Sulfuro de Hidrógeno/uso terapéutico , Ligadura/efectos adversos , Flujo Sanguíneo Regional/efectos de los fármacos , Choque Séptico/tratamiento farmacológico , Animales , Sulfuro de Hidrógeno/farmacología , Inflamación/sangre , Inflamación/tratamiento farmacológico , Inflamación/etiología , Inflamación/inmunología , Interleucina-5/sangre , Interleucina-6/sangre , Masculino , Infiltración Neutrófila/fisiología , Punciones/efectos adversos , Ratas , Ratas Sprague-Dawley , Choque Séptico/sangre , Choque Séptico/etiología , Choque Séptico/inmunología , Factores de Tiempo

RESUMEN

Mitochondria are organelles which, according to the endosymbiosis theory, evolved from purpurbacteria approximately 1.5 billion years ago. One of the unique features of mitochondria is that they have their own genome. Mitochondria replicate and transcribe their DNA semiautonomously. Like nuclear DNA, mitochondrial DNA (mtDNA) is constantly exposed to DNA damaging agents. Regarding the repair of mtDNA, the prevailing concept for many years was that mtDNA molecules suffering an excess of damage would simply be degraded to be replaced by newly generated successors copied from undamaged genomes. However, evidence now clearly shows that mitochondria contain the machinery to repair the damage to their genomes caused by certain endogenous or exogenous damaging agents. The link between mtDNA damage and repair to aging, neurodegeneration, and carcinogenesis-associated processes is the subject of this review.

Asunto(s)

Envejecimiento/genética , Reparación del ADN , ADN Mitocondrial/metabolismo , Genoma Mitocondrial , Neoplasias/genética , Enfermedades Neurodegenerativas/genética , Daño del ADN , Humanos , Mutación

RESUMEN

Primary astrocyte cultures were used to investigate the modulation of DNA repair as a tool for sensitizing astrocytes to genotoxic agents. Base excision repair (BER) is the principal mechanism by which mammalian cells repair alkylation damage to DNA and involves the processing of relatively nontoxic DNA adducts through a series of cytotoxic intermediates during the course of restoring normal DNA integrity. An adenoviral expression system was employed to target high levels of the BER pathway initiator, N-methylpurine glycosylase (MPG), to either the mitochondria or nucleus of primary astrocytes to test the hypothesis that an alteration in BER results in increased alkylation sensitivity. Increasing MPG activity significantly increased BER kinetics in both the mitochondria and nuclei. Although modulating MPG activity in mitochondria appeared to have little effect on alkylation sensitivity, increased nuclear MPG activity resulted in cell death in astrocyte cultures treated with methylnitrosourea (MNU). Caspase-3 cleavage was not detected, thus indicating that these alkylation sensitive astrocytes do not undergo a typical programmed cell death in response to MNU. Astrocytes were found to express relatively high levels of antiapoptotic Bcl-2 and Bcl-XL and very low levels of proapoptotic Bad and Bid suggesting that the mitochondrial pathway of apoptosis may be blocked making astrocytes less vulnerable to proapoptotic stimuli compared with other cell types. Consequently, this unique characteristic of astrocytes may be responsible, in part, for resistance of astrocytomas to chemotherapeutic agents.

Asunto(s)

Antineoplásicos/farmacología , Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , ADN Glicosilasas/farmacología , Reparación del ADN/genética , Resistencia a Antineoplásicos/genética , Alquilación/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Astrocitos/metabolismo , Disparidad de Par Base/genética , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Núcleo Celular/metabolismo , Células Cultivadas , Daño del ADN/efectos de los fármacos , Daño del ADN/genética , ADN Glicosilasas/uso terapéutico , Reparación del ADN/efectos de los fármacos , Metilnitrosourea/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/genética , Mitocondrias/metabolismo , Mutágenos/farmacología , Ratas , Ratas Sprague-Dawley

RESUMEN

Nitric oxide (NO) that is produced by inducible NO synthase (iNOS) in glial cells is thought to contribute significantly to the pathogenesis of multiple sclerosis. Oligodendrocytes can be stimulated to express iNOS by inflammatory cytokines, which are known to accumulate in the multiple sclerotic brain. The potentially pathological levels of NO produced under these circumstances can target a wide spectrum of intracellular components. We hypothesized that one of the critical targets for damage that leads to disease is mtDNA. In this study, we found that cytokines, in particular a combination of tumor necrosis factor-alpha (50 ng/ml) and IFNgamma (25 ng/ml), cause elevated NO production in primary cultures of rat oligodendrocytes. Western blot analysis revealed a strong enhancement of iNOS expression 48 h after cytokine treatment. Within the same time period, NO-mediated mtDNA damage was shown by Southern blot analysis and by ligation-mediated PCR. Targeting the DNA repair enzyme human 8-oxoguanine DNA glycosylase (hOGG1) to the mitochondria of oligodendrocytes had a protective effect against this cytokine-mediated mtDNA damage. Moreover, it was shown that mitochondrial transport sequence hOGG1-transfected oligodendrocytes had fewer apoptotic cells compared with cells containing vector only following treatment with the cytokines. Subsequent experiments revealed that targeting hOGG1 to mitochondria reduces the activation of caspase-9, showing that this recombinant protein works to reduce apoptosis that is occurring through a mitochondria-based pathway.

Asunto(s)

Apoptosis , Citocinas/química , ADN Glicosilasas/química , ADN Mitocondrial/química , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Animales , Western Blotting , Encéfalo/metabolismo , Caspasa 8 , Caspasa 9 , Caspasas/metabolismo , Supervivencia Celular , Citocinas/metabolismo , Citosol/metabolismo , Daño del ADN , ADN Mitocondrial/metabolismo , Electroforesis en Gel de Poliacrilamida , Humanos , Inflamación , Interferón gamma/metabolismo , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/patología , Neuroglía/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo II , Oligodendroglía/metabolismo , ARN/metabolismo , ARN Mitocondrial , Ratas , Proteínas Recombinantes/química , Factores de Tiempo , Transfección , Factor de Necrosis Tumoral alfa/metabolismo

RESUMEN

Within the central nervous system (CNS), there is a differential susceptibility among cell types to certain pathological conditions believed to involve oxidative stress. Oligodendrocytes are extremely sensitive to oxidative stress, which correlates with a decreased ability to repair damage in mitochondrial DNA (mtDNA), as we have shown previously. To determine whether there is a causal relationship, studies were carried out to correct the deficit in repair of the oxidative damage in mtDNA in cultured oligodendrocytes. A vector containing a mitochondrial transport sequence (MTS) upstream of the sequence for human 8-oxoguanine-DNA glycosylase (OGG) was transfected into the cells. The efficiency of transfection and the localization of recombinant protein were determined by fluorescence microscopy and by Western blot analysis. Subsequent mtDNA repair studies, employing 100 micro M menadione to produce reactive oxygen species, showed a significant enhancement in repair of oxidative lesions in mtDNA of MTS-OGG transfected oligodendrocytes compared with cells transfected with vector only. Experiments were also conducted to determine the effect of changing mtDNA repair capacity on menadione-induced apoptosis in oligodendrocytes. These experiments show that targeting the OGG repair enzyme to mitochondria reduces the release of cytochrome c from the intermitochondrial space and the activation of caspase 9 in oligodendrocytes after exposure to menadione. Therefore, targeting of DNA repair enzymes to mitochondria appears to be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.

Asunto(s)

Mitocondrias/enzimología , N-Glicosil Hidrolasas/genética , N-Glicosil Hidrolasas/metabolismo , Oligodendroglía/enzimología , Estrés Oxidativo/fisiología , Animales , Antifibrinolíticos/farmacología , Apoptosis/fisiología , Células Cultivadas , Daño del ADN/fisiología , Reparación del ADN/fisiología , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , ADN-Formamidopirimidina Glicosilasa , Humanos , Oligodendroglía/citología , Estrés Oxidativo/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismo , Transfección/métodos , Vitamina K 3/farmacología