RESUMEN
With the increasing use of vaping devices that deliver high levels of nicotine (NIC) to the lungs, sporadic lung injury has been observed. Commercial vaping solutions can contain high NIC concentrations of 150 mM or more. With high NIC levels, its metabolic products may induce toxicity. NIC is primarily metabolized to form NIC iminium (NICI) which is further metabolized by aldehyde oxidase (AOX) to cotinine. We determine that NICI in the presence of AOX is a potent trigger of superoxide generation. NICI stimulated superoxide generation from AOX with Km = 2.7 µM and Vmax = 794 nmol/min/mg measured by cytochrome-c reduction. EPR spin-trapping confirmed that NICI in the presence of AOX is a potent source of superoxide. AOX is expressed in the lungs and chronic e-cigarette exposure in mice greatly increased AOX expression. NICI or NIC stimulated superoxide production in the lungs of control mice with an even greater increase after chronic e-cigarette exposure. This superoxide production was quenched by AOX inhibition. Furthermore, e-cigarette-mediated NIC delivery triggered oxidative lung damage that was blocked by AOX inhibition. Thus, NIC metabolism triggers AOX-mediated superoxide generation that can cause lung injury. Therefore, high uncontrolled levels of NIC inhalation, as occur with e-cigarette use, can induce oxidative lung damage.
RESUMEN
Cytoglobin (Cygb) was discovered as a novel type of globin that is expressed in mammals; however, its functions remain uncertain. While Cygb protects against oxidant stress, the basis for this is unclear, and the effect of Cygb on superoxide metabolism is unknown. From dose-dependent studies of the effect of Cygb on superoxide catabolism, we identify that Cygb has potent superoxide dismutase (SOD) function. Initial assays using cytochrome c showed that Cygb exhibits a high rate of superoxide dismutation on the order of 108 M-1 â s-1 Spin-trapping studies also demonstrated that the rate of Cygb-mediated superoxide dismutation (1.6 × 108 M-1 â s-1) was only â¼10-fold less than Cu,Zn-SOD. Stopped-flow experiments confirmed that Cygb rapidly dismutates superoxide with rates within an order of magnitude of Cu,Zn-SOD or Mn-SOD. The SOD function of Cygb was inhibited by cyanide and CO that coordinate to Fe3+-Cygb and Fe2+-Cygb, respectively, suggesting that dismutation involves iron redox cycling, and this was confirmed by spectrophotometric titrations. In control smooth-muscle cells and cells with siRNA-mediated Cygb knockdown subjected to extracellular superoxide stress from xanthine/xanthine oxidase or intracellular superoxide stress triggered by the uncoupler, menadione, Cygb had a prominent role in superoxide metabolism and protected against superoxide-mediated death. Similar experiments in vessels showed higher levels of superoxide in Cygb-/- mice than wild type. Thus, Cygb has potent SOD function and can rapidly dismutate superoxide in cells, conferring protection against oxidant injury. In view of its ubiquitous cellular expression at micromolar concentrations in smooth-muscle and other cells, Cygb can play an important role in cellular superoxide metabolism.
Asunto(s)
Citoglobina , Superóxido Dismutasa , Animales , Línea Celular , Citoglobina/química , Citoglobina/genética , Citoglobina/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Masculino , Ratones , Ratones Noqueados , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/química , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
In smooth muscle, cytoglobin (Cygb) functions as a potent nitric oxide (NO) dioxygenase and regulates NO metabolism and vascular tone. Major questions remain regarding which cellular reducing systems regulate Cygb-mediated NO metabolism. To better define the Cygb-mediated NO dioxygenation process in vascular smooth muscle cells (SMCs), and the requisite reducing systems that regulate cellular NO decay, we assessed the intracellular concentrations of Cygb and its putative reducing systems and examined their roles in the process of NO decay. Cygb and the reducing systems, cytochrome b5 (B5)/cytochrome b5 reductase (B5R) and cytochrome P450 reductase (CPR) were measured in aortic SMCs. Intracellular Cygb concentration was estimated as 3.5 µM, while B5R, B5, and CPR were 0.88, 0.38, and 0.15 µM, respectively. NO decay in SMCs was measured following bolus addition of NO to air-equilibrated cells. siRNA-mediated knockdown experiments indicated that â¼78% of NO metabolism in SMCs is Cygb-dependent. Of this, â¼87% was B5R- and B5-dependent. CPR knockdown resulted in a small decrease in the NO dioxygenation rate (VNO), while depletion of ascorbate had no effect. Kinetic analysis of VNO for the B5/B5R/Cygb system with variation of B5 or B5R concentrations from their SMC levels showed that VNO exhibits apparent Michaelis-Menten behavior for B5 and B5R. In contrast, linear variation was seen with change in Cygb concentration. Overall, B5/B5R was demonstrated to be the major reducing system supporting Cygb-mediated NO metabolism in SMCs with changes in cellular B5/B5R levels modulating the process of NO decay.
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Citocromos b5/metabolismo , Citoglobina/metabolismo , Músculo Liso Vascular/metabolismo , Óxido Nítrico/metabolismo , Oxigenasas/metabolismo , Animales , Fenómenos Bioquímicos , Células Cultivadas , Humanos , Cinética , RatonesRESUMEN
We recently reported a mouse model of chronic electronic cigarette (e-cig) exposure-induced cardiovascular pathology, where long-term exposure to e-cig vape (ECV) induces cardiac abnormalities, impairment of endothelial function, and systemic hypertension. Here, we delineate the underlying mechanisms of ECV-induced vascular endothelial dysfunction (VED), a central trigger of cardiovascular disease. C57/BL6 male mice were exposed to ECV generated from e-cig liquid containing 0, 6, or 24 mg/mL nicotine for 16 and 60 wk. Time-dependent elevation in blood pressure and systemic vascular resistance were observed, along with an impairment of acetylcholine-induced aortic relaxation in ECV-exposed mice, compared with air-exposed control. Decreased intravascular nitric oxide (NO) levels and increased superoxide generation with elevated 3-nitrotyrosine levels in the aorta of ECV-exposed mice were observed, indicating that ECV-induced superoxide reacts with NO to generate cytotoxic peroxynitrite. Exposure increased NADPH oxidase expression, supporting its role in ECV-induced superoxide generation. Downregulation of endothelial nitric oxide synthase (eNOS) expression and Akt-dependent eNOS phosphorylation occurred in the aorta of ECV-exposed mice, indicating that exposure inhibited de novo NO synthesis. Following ECV exposure, the critical NOS cofactor tetrahydrobiopterin was decreased, with a concomitant loss of its salvage enzyme, dihydrofolate reductase. NADPH oxidase and NOS inhibitors abrogated ECV-induced superoxide generation in the aorta of ECV-exposed mice. Together, our data demonstrate that ECV exposure activates NADPH oxidase and uncouples eNOS, causing a vicious cycle of superoxide generation and vascular oxidant stress that triggers VED and hypertension with predisposition to other cardiovascular disease.NEW & NOTEWORTHY Underlying mechanisms of e-cig-induced vascular endothelial dysfunction are delineated. e-cig exposure activates and increases expression of NADPH oxidase and disrupts activation and coupling of eNOS, leading to a vicious cycle of superoxide generation and peroxynitrite formation, with tetrahydrobiopterin depletion, causing loss of NO that triggers vascular endothelial dysfunction. This process is progressive, increasing with the duration of e-cig exposure, and is more severe in the presence of nicotine, but observed even with nicotine-free vaping.
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Enfermedades Cardiovasculares , Sistemas Electrónicos de Liberación de Nicotina , Hipertensión , Animales , Endotelio Vascular/metabolismo , Femenino , Masculino , Ratones , NADPH Oxidasas/metabolismo , Nicotina , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ácido Peroxinitroso/metabolismo , Superóxidos/metabolismoRESUMEN
Cytoglobin (Cygb) has been identified as the major nitric oxide (NO) metabolizing protein in vascular smooth muscle cells (VSMCs) and is crucial for the regulation of vascular tone. In the presence of its requisite cytochrome B5a (B5)/B5 reductase-isoform-3 (B5R) reducing system, Cygb controls NO metabolism through the oxygen-dependent process of NO dioxygenation. Tobacco cigarette smoking (TCS) induces vascular dysfunction; however, the role of Cygb in the pathophysiology of TCS-induced cardiovascular disease has not been previously investigated. While TCS impairs NO biosynthesis, its effect on NO metabolism remains unclear. Therefore, we performed studies in aortic VSMCs with tobacco smoke extract (TSE) exposure to investigate the effects of cigarette smoke constituents on the rates of NO decay, with focus on the alterations that occur in the process of Cygb-mediated NO metabolism. TSE greatly enhanced the rates of NO metabolism by VSMCs. An initial increase in superoxide-mediated NO degradation was seen at 4 h of exposure. This was followed by much larger progressive increases at 24 and 48 h, accompanied by parallel increases in the expression of Cygb and B5/B5R. siRNA-mediated Cygb knockdown greatly decreased these TSE-induced elevations in NO decay rates. Therefore, upregulation of the levels of Cygb and its reducing system accounted for the large increase in NO metabolism rate seen after 24 h of TSE exposure. Thus, increased Cygb-mediated NO degradation would contribute to TCS-induced vascular dysfunction and partial inhibition of Cygb expression or its NO dioxygenase function could be a promising therapeutic target to prevent secondary cardiovascular disease.
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Citoglobina/metabolismo , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Contaminación por Humo de Tabaco/efectos adversos , Animales , Aorta/citología , Supervivencia Celular/efectos de los fármacos , Citocromo-B(5) Reductasa/metabolismo , Citocromos b5/metabolismo , Citoglobina/genética , Técnicas de Silenciamiento del Gen , Ratones , Músculo Liso Vascular/citología , Superóxidos/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Electronic cigarette (e-cig) vaping (ECV) has been proposed as a safer alternative to tobacco cigarette smoking (TCS); however, this remains controversial due to a lack of long-term comparative studies. Therefore, we developed a chronic mouse exposure model that mimics human vaping and allows comparison with TCS. Longitudinal studies were performed to evaluate alterations in cardiovascular function with TCS and ECV exposure durations of up to 60 wk. For ECV, e-cig liquid with box-mod were used and for TCS, 3R4F-cigarettes. C57/BL6 male mice were exposed 2 h/day, 5 days/wk to TCS, ECV, or air control. The role of vape nicotine levels was evaluated using e-cig-liquids with 0, 6, or 24 mg/mL nicotine. Following 16-wk exposure, increased constriction to phenylephrine and impaired endothelium-dependent and endothelium-independent vasodilation were observed in aortic segents, paralleling the onset of systemic hypertension, with elevations in systemic vascular resistance. Following 32 wk, TCS and ECV induced cardiac hypertrophy. All of these abnormalities further increased out to 60 wk of exposure, with elevated heart weight and aortic thickness along with increased superoxide production in vessels and cardiac tissues of both ECV and TCS mice. While ECV-induced abnormalities were seen in the absence of nicotine, these occurred earlier and were more severe with higher nicotine exposure. Thus, long-term vaping of e-cig can induce cardiovascular disease similar to TCS, and the severity of this toxicity increases with exposure duration and vape nicotine content.NEW & NOTEWORTHY A chronic mouse exposure model that mimics human e-cigarette vaping and allows comparison with tobacco cigarette smoking was developed and utilized to perform longitudinal studies of alterations in cardiovascular function. E-cigarette exposure led to the onset of cardiovascular disease similar to that with tobacco cigarette smoking. Impaired endothelium-dependent and endothelium-independent vasodilation with increased adrenergic vasoconstriction were observed, paralleling the onset of systemic hypertension and subsequent cardiac hypertrophy. This cardiovascular toxicity was dependent on exposure duration and nicotine dose.
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Aorta/efectos de los fármacos , Enfermedades Cardiovasculares/inducido químicamente , Nicotina/administración & dosificación , Vapeo/efectos adversos , Agonistas de Receptores Adrenérgicos alfa 1/farmacología , Animales , Aorta/fisiopatología , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Enfermedades Cardiovasculares/fisiopatología , Sistemas Electrónicos de Liberación de Nicotina , Masculino , Ratones , Fenilefrina/farmacología , Factores de Tiempo , Vasodilatación/efectos de los fármacos , Vasodilatación/fisiologíaRESUMEN
Although there is a strong association between cigarette smoking exposure (CSE) and vascular endothelial dysfunction (VED), the underlying mechanisms by which CSE triggers VED remain unclear. Therefore, studies were performed to define these mechanisms using a chronic mouse model of cigarette smoking (CS)-induced cardiovascular disease mirroring that in humans. C57BL/6 male mice were subjected to CSE for up to 48 wk. CSE impaired acetylcholine (ACh)-induced relaxation of aortic and mesenteric segments and triggered hypertension, with mean arterial blood pressure at 32 and 48 wk of exposure of 122 ± 6 and 135 ± 5 mmHg compared with 99 ± 4 and 102 ± 6 mmHg, respectively, in air-exposed mice. CSE led to monocyte activation with superoxide generation in blood exiting the pulmonary circulation. Macrophage infiltration with concomitant increase in NADPH oxidase subunits p22phox and gp91phox was seen in aortas of CS-exposed mice at 16 wk, with further increase out to 48 wk. Associated with this, increased superoxide production was detected that decreased with Nox inhibition. Tetrahydrobiopterin was progressively depleted in CS-exposed mice but not in air-exposed controls, resulting in endothelial nitric oxide synthase (eNOS) uncoupling and secondary superoxide generation. CSE led to a time-dependent decrease in eNOS and Akt expression and phosphorylation. Overall, CSE induces vascular monocyte infiltration with increased NADPH oxidase-mediated reactive oxygen species generation and depletes the eNOS cofactor tetrahydrobiopterin, uncoupling eNOS and triggering a vicious cycle of oxidative stress with VED and hypertension. Our study provides important insights toward understanding the process by which smoking contributes to the genesis of cardiovascular disease and identifies biomarkers predictive of disease.NEW & NOTEWORTHY In a chronic model of smoking-induced cardiovascular disease, we define underlying mechanisms of smoking-induced vascular endothelial dysfunction (VED). Smoking exposure triggered VED and hypertension and led to vascular macrophage infiltration with concomitant increase in superoxide and NADPH oxidase levels as early as 16 wk of exposure. This oxidative stress was accompanied by tetrahydrobiopterin depletion, resulting in endothelial nitric oxide synthase uncoupling with further superoxide generation triggering a vicious cycle of oxidative stress and VED.
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Endotelio Vascular/metabolismo , Leucocitos/metabolismo , Estrés Oxidativo , Lesión por Inhalación de Humo/metabolismo , Contaminación por Humo de Tabaco/efectos adversos , Vasodilatación , Animales , Aorta/metabolismo , Aorta/fisiopatología , Presión Sanguínea , Endotelio Vascular/fisiopatología , Masculino , Arterias Mesentéricas/metabolismo , Arterias Mesentéricas/fisiopatología , Ratones , Ratones Endogámicos C57BL , NADPH Oxidasas/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Lesión por Inhalación de Humo/etiología , Lesión por Inhalación de Humo/fisiopatología , Superóxidos/metabolismoRESUMEN
Objectives: To develop and test a new system for whole body exposure of small animals to support investigation of the biological effects of aerosol generated by electronic cigarette (e-cig) products under diverse inhalation conditions with improved control and monitoring of the e-cig vape exposure and nicotine delivered to the animal's systemic circulation. Methods: A computer-controlled design, with built-in sensors for real time monitoring of O2, CO2, relative humidity, and temperature within the exposure chambers and port for measuring total particulate matter (TPM) was developed, constructed and tested. This design accommodates a variety of commercial vaping devices, offers software flexibility to adjust exposure protocols to mimic different users' puffing patterns, enables variable nicotine delivery to the animal's systemic circulation; minimizes travel time and alterations of aerosol quality or quantity by delivering aerosol directly to the exposure chamber, offers local or remote operation of up to six distinct exposure chambers from a single control unit, and can simultaneously test different exposure conditions or products in diverse animal groups, which reduces inter-run variability, saves time, and increases productivity. Results: The time course pattern of TPM concentration during different phases of the exposure cycle was measured. With increased puffing duration or number of exposure cycles, higher TPM exposure and plasma cotinine levels were observed with plasma cotinine levels in the range reported in light or heavy smokers. Conclusion: Overall, this novel, versatile, and durable exposure system facilitates high-throughput evaluation of the relative safety and potential toxicity of a variety of e-cig devices and liquids.
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Sistemas Electrónicos de Liberación de Nicotina , Pruebas de Toxicidad/instrumentación , Administración por Inhalación , Animales , Dióxido de Carbono/análisis , Monóxido de Carbono/análisis , Cotinina/sangre , Diseño de Equipo , Humedad , Masculino , Ratones Endogámicos C57BL , Oxígeno/análisis , Material Particulado/análisis , Material Particulado/toxicidad , TemperaturaRESUMEN
While chronic smoking triggers cardiovascular disease, controversy remains regarding its effects on the brain and cognition. We investigated the effects of long-term cigarette smoke (CS) exposure (CSE) on cerebrovascular function, neuronal injury, and cognition in a novel mouse exposure model. Longitudinal studies were performed in CS or air-exposed mice, 2 hours/day, for up to 60 weeks. Hypertension and carotid vascular endothelial dysfunction (VED) occurred by 16 weeks of CSE, followed by reduced carotid artery blood flow, with oxidative stress detected in the carotid artery, and subsequently in the brain of CS-exposed mice with generation of reactive oxygen species (ROS) and secondary protein and DNA oxidation, microglial activation and astrocytosis. Brain small vessels exhibited decreased levels of endothelial NO synthase (eNOS), enlarged perivascular spaces with blood brain barrier (BBB) leak and decreased levels of tight-junction proteins. In the brain, amyloid-ß deposition and phosphorylated-tau were detected with increases out to 60 weeks, at which time mice exhibited impaired spatial learning and memory. Thus, long-term CSE initiates a cascade of ROS generation and oxidative damage, eNOS dysfunction with cerebral hypoperfusion, as well as cerebrovascular and BBB damage with intracerebral inflammation, and neuronal degeneration, followed by the onset of impaired cognition and memory.
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Cytoglobin (Cygb) is a recently discovered cytoplasmic heme-binding globin. Although multiple hemeproteins have been reported to function as nitrite reductases in mammalian cells, it is unknown whether Cygb can also reduce nitrite to nitric oxide (NO). The mechanism, magnitude, and quantitative importance of Cygb-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, spectrophotometric measurements, and chemiluminescence NO analyzer studies were performed. Under anaerobic conditions, mixing nitrite with ferrous-Cygb triggered NO formation that was trapped and detected using EPR spin trapping. Spectrophotometric studies revealed that nitrite binding to ferrous-Cygb is followed by formation of ferric-Cygb and NO. The kinetics and magnitude of Cygb-mediated NO formation were characterized. It was observed that Cygb-mediated NO generation increased linearly with the increase of nitrite concentration under anaerobic conditions. This Cygb-mediated NO production greatly increased with acidosis and near-anoxia as occur in ischemic conditions. With the addition of nitrite, soluble guanylyl cyclase activation was significantly higher in normal smooth muscle cells compared with Cygb knocked down cells with Cygb accounting for â¼40% of the activation in control cells and â¼60% in cells subjected to hypoxia for 48 h. Overall, these studies show that Cygb-mediated nitrite reduction can play an important role in NO generation and soluble guanylyl cyclase activation under hypoxic conditions, with this process regulated by pH, oxygen tension, nitrite concentration, and the redox state of the cells.
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Globinas/metabolismo , Miocitos del Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Nitritos/metabolismo , Anaerobiosis , Hipoxia de la Célula/fisiología , Células Cultivadas , Citoglobina , Espectroscopía de Resonancia por Spin del Electrón , Globinas/química , Globinas/genética , Guanilato Ciclasa/química , Guanilato Ciclasa/genética , Guanilato Ciclasa/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Cinética , Mediciones Luminiscentes , Miocitos del Músculo Liso/citología , Óxido Nítrico/química , Nitritos/química , Oxidación-Reducción , Oxígeno/química , Oxígeno/metabolismoRESUMEN
PURPOSE: This study was designed to investigate the effect of intramarrow penetration (IMP) and 1% melatonin (MLN) gel on the remodelling process of autogenous bone graft (ABG) in an induced 1-osseous wall defect model. METHODS: Sixty-four intrabony induced mandibular defects were created on the distal side of premolars-P1, P2, P3, and P4 (on each side)-in 8 beagle dogs. A ligature-induced periodontitis was initiated in each defect. Defects were then divided into 4 equal groups. Group I was treated with open-flap debridement (OFD) alone, group II was treated with OFD/ABG, group III was treated with OFD/IMP/ABG, and group IV was treated with OFD/ABG/IMP/1% MLN gel. The study parameters were bone fill, histologic analysis, and immunohistochemical evaluation of endothelial nitric oxide synthase (eNOS) expression at 2-week (2W) and 8-week (8W) time intervals. RESULTS: At 8W, significant differences were revealed amongst all groups regarding the amount of bone fill and eNOS expressions (P < .001). Bone fill percentages were 55.5%, 22.3%, 16.8%, and 0% in groups IV, III, II, and I, respectively. eNOS expressions were 1.68 ± 0.06, 8.43 ± 0.04, 16.80 ± 0.17, and 1.97 ± 0.07 in groups IV, III, II, and I, respectively. The favourable results were in line with group IV. CONCLUSIONS: According to these preliminary results, defects treated by ABG augmented with IMP and 1% MLN gel revealed a greater amount of bone fill and reduced eNOS expression. This combination is therefore highly suggested as an adjunct to ABG.
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Pérdida de Hueso Alveolar , Melatonina , Perros , Humanos , Animales , Pérdida de Hueso Alveolar/cirugía , Melatonina/farmacología , Melatonina/uso terapéutico , Regeneración Tisular Guiada Periodontal , Resultado del TratamientoRESUMEN
Cigarette smoke exposure is known to induce obstructive lung disease and several cardiovascular disease states in humans and also in animal models. Smoking leads to oxidative stress and inflammation that are important in triggering pulmonary and cardiovascular disease. The objective of the current study was to quantify differences in expression levels of plasma proteins of cigarette smoke -exposed and control mice, at the time of disease onset, and identify these proteins for use as potential biomarkers of the onset of smoking-induced disease. We utilized 2-D DIGE/MS to characterize these proteomic changes. 2-D DIGE of plasma samples identified 11 differentially expressed proteins in cigarette smoke -exposed mice. From these 11 proteins, 9 were downregulated and 2 were upregulated. The proteins identified are involved in vascular function, coagulation, metabolism and immune function. Among these, the alterations in fibrinogen (2.2-fold decrease), α-1-antitrypsin (1.8-fold increase) and arginase (4.5-fold decrease) are of particular interest since these have been directly linked to cardiovascular and lung pathology. Differences in expression levels of these proteins were also confirmed by immunoblotting. Thus, we observe that chronic cigarette smoke exposure in mice leads to prominent changes in the protein expression profile of blood plasma and these changes in turn can potentially serve as markers predictive of the onset and progression of cardiovascular and pulmonary disease.
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Proteínas Sanguíneas/análisis , Electroforesis en Gel Bidimensional/métodos , Espectrometría de Masas/métodos , Proteoma/química , Contaminación por Humo de Tabaco , Animales , Biomarcadores , Proteínas Sanguíneas/metabolismo , Western Blotting , Carbocianinas , Estudios de Casos y Controles , Procesamiento de Imagen Asistido por Computador , Masculino , Redes y Vías Metabólicas , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Proteoma/metabolismo , Transducción de SeñalRESUMEN
Cigarette smoking is a major independent risk factor for cardiovascular disease. While the association between chronic smoking and cardiovascular disease is well established, the underlying mechanisms are incompletely understood, partly due to the lack of adequate in vivo animal models. Here, we report a mouse model of chronic smoking-induced cardiovascular pathology. Male C57BL/6J mice were exposed to whole body mainstream cigarette smoke (CS) using a SCIREQ "InExpose" smoking system (48 min/day, 5 days/wk) for 16 or 32 wk. Age-matched, air-exposed mice served as nonsmoking controls. Blood pressure was measured, and cardiac MRI was performed. In vitro vascular ring and isolated heart experiments were performed to measure vascular reactivity and cardiac function. Blood from control and smoking mice was studied for the nitric oxide (NO) decay rate and reactive oxygen species (ROS) generation. With 32 wk of CS exposure, mice had significantly less body weight gain and markedly higher blood pressure. At 32 wk of CS exposure, ACh-induced vasorelaxation was significantly shifted to the right and downward, left ventricular mass was significantly larger along with an increased heart-to-body weight ratio, in vitro cardiac function tended to be impaired with high afterload, white blood cells had significantly higher ROS generation, and the blood NO decay rate was significantly faster. Thus, smoking led to blunted weight gain, hypertension, endothelial dysfunction, leukocyte activation with ROS generation, decreased NO bioavailability, and mild cardiac hypertrophy in mice that were not otherwise predisposed to disease. This mouse model is a useful tool to enable further elucidation of the molecular and cellular mechanisms of smoking-induced cardiovascular diseases.
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Endotelio Vascular/fisiopatología , Hipertensión/etiología , Óxido Nítrico/metabolismo , Estrés Oxidativo , Fumar/efectos adversos , Remodelación Ventricular , Análisis de Varianza , Animales , Presión Sanguínea , Peso Corporal , Endotelio Vascular/metabolismo , Corazón/fisiopatología , Hipertensión/metabolismo , Imagen por Resonancia Magnética , Masculino , Ratones , Miocardio/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Riesgo , Humo , NicotianaRESUMEN
The use of innocuous naturally occurring compounds to overcome drug resistance and cancer recalcitrance is now in the forefront of cancer research. Thymoquinone (TQ) is a bioactive constituent of the volatile oil derived from seeds of Nigella sativa Linn. TQ has shown promising anti-carcinogenic and anti-tumor activities through different mechanisms. However, the effect of TQ on cell signaling and survival pathways in resistant cancer cells has not been fully delineated. Here, we report that TQ greatly inhibits doxorubicin-resistant human breast cancer MCF-7/DOX cell proliferation. TQ treatment increased cellular levels of PTEN proteins, resulting in a substantial decrease of phosphorylated Akt, a known regulator of cell survival. The PTEN expression was accompanied with elevation of PTEN mRNA. TQ arrested MCF-7/DOX cells at G2/M phase and increased cellular levels of p53 and p21 proteins. Flow cytometric analysis and agarose gel electrophoresis revealed a significant increase in Sub-G1 cell population and appearance of DNA ladders following TQ treatment, indicating cellular apoptosis. TQ-induced apoptosis was associated with disrupted mitochondrial membrane potential and activation of caspases and PARP cleavage in MCF-7/DOX cells. Moreover, TQ treatment increased Bax/Bcl2 ratio via up-regulating Bax and down-regulating Bcl2 proteins. More importantly, PTEN silencing by target specific siRNA enabled the suppression of TQ-induced apoptosis resulting in increased cell survival. Our results reveal that up-regulation of the key upstream signaling factor, PTEN, in MCF-7/DOX cells inhibited Akt phosphorylation, which ultimately causes increase in their regulatory p53 levels affecting the induction of G2/M cell cycle arrest and apoptosis. Overall results provide mechanistic insights for understanding the molecular basis and utility of the anti-tumor activity of TQ.
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Apoptosis/efectos de los fármacos , Benzoquinonas/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Fosfohidrolasa PTEN/metabolismo , Benzoquinonas/química , Western Blotting , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Caspasas/metabolismo , División Celular/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fragmentación del ADN/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Doxorrubicina/farmacología , Femenino , Fase G2/efectos de los fármacos , Humanos , Estructura Molecular , Fosfohidrolasa PTEN/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Restoration of functionally intact chromatin structure following DNA damage processing is crucial for maintaining genetic and epigenetic information in human cells. Here, we show the UV-induced uH2A foci formation in cells lacking XPC, DDB2, CSA or CSB, but not in cells lacking XPA, XPG or XPF indicating that uH2A incorporation relied on successful damage repair occurring through either GGR or TCR sub-pathway. In contrast, XPA, XPG or XPF were not required for formation of gammaH2AX foci in asynchronous cells. Notably, the H2A ubiquitin ligase Ring1B, a component of Polycomb repressor complex 1, did not localize at DNA damage sites. However, histone chaperone CAF-1 showed distinct localization to the damage sites. Knockdown of CAF-1 p60 abolished CAF-1 as well as uH2A foci formation. CAF-1 p150 was found to associate with NER factors TFIIH, RPA p70 and PCNA in chromatin. These data demonstrate that successful NER of genomic lesions and prompt CAF-1-mediated chromatin restoration link uH2A incorporation at the sites of damage repair within chromatin.
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Cromatina/metabolismo , Daño del ADN , Reparación del ADN , Genoma Humano/genética , Histonas/metabolismo , Ubiquitinación , Proteínas de Ciclo Celular/metabolismo , Factor 1 de Ensamblaje de la Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Complejo Represivo Polycomb 1 , Antígeno Nuclear de Célula en Proliferación/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Transporte de Proteínas , Proteína de Replicación A/metabolismo , Factores de Transcripción , Transcripción Genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinas/metabolismoRESUMEN
Cisplatin is one of the most widely used anticancer agents, displaying activity against a wide variety of tumors. However, development of drug resistance presents a challenging barrier to successful cancer treatment by cisplatin. To understand the mechanism of cisplatin resistance, we investigated the role of damaged DNA binding protein complex subunit 2 (DDB2) in cisplatin-induced cytotoxicity and apoptosis. We show that DDB2 is not required for the repair of cisplatin-induced DNA damage, but can be induced by cisplatin treatment. DDB2-deficient noncancer cells exhibit enhanced resistance to cell growth inhibition and apoptosis induced by cisplatin than cells with fully restored DDB2 function. Moreover, DDB2 expression in cisplatin-resistant ovarian cancer cell line CP70 and MCP2 was lower than their cisplatin-sensitive parental A2780 cells. Overexpression of DDB2 sensitized CP70 cells to cisplatin-induced cytotoxicity and apoptosis via activation of the caspase pathway and downregulation of antiapoptotic Bcl-2 protein. Further analysis indicates that the overexpression of DDB2 in CP70 cells downregulates Bcl-2 expression through decreasing Bcl-2 mRNA level. These results suggest that ovarian cancer cells containing high level of DDB2 become susceptible to cisplatin by undergoing enhanced apoptosis.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cisplatino/farmacología , Proteínas de Unión al ADN/metabolismo , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/genética , Femenino , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Neoplasias Ováricas/tratamiento farmacológico , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
While radiotherapy is a widely used treatment for many types of human cancer, problems of radio-resistance and side effects remain. Side effects induced by ionizing radiation (IR) arise primarily from its propensity to trigger inflammation and oxidative stress with damage of normal cells and tissues near the treatment area. The highly potent superoxide dismutase mimetic, GC4419 (Galera Therapeutics), rapidly enters cells and is highly effective in dismutating superoxide (O2â¢-). We performed studies to assess the potency of GC4419 in cancer killing and radio-sensitization in human lung cancer cells and normal immortalized lung cells. Treatment with GC4419 did not alter the radical generation during IR, primarily hydroxyl radical (.OH); however, it quenched the increased levels of O2â¢- detected in the cancer cells before and following IR. GC4419 triggered cancer cell death and inhibited cancer cell proliferation with no adverse effect on normal cells. Combination of GC4419 with IR augmented the cytotoxic effects of IR on cancer cells compared to monotherapy, while protecting normal cells from IR-induced cell death. DNA fragmentation and caspase-3 activity assays showed that combination of GC4419 with IR enhances cancer cell apoptosis. Moreover, GC4419 increased IR-induced Bax levels with decreased Bcl-2 and elevated Bax/Bcl-2 ratio following treatment. GC4419 increased TrxR activity in the normal cells but decreased activity in cancer cells, conferring increased cancer cell sensitivity to oxidative stress. In conclusion, GC4419 increases the cytotoxic and pro-apoptotic activity of IR in lung cancer cells while decreasing injury in normal cells.
Asunto(s)
Neoplasias , Compuestos Organometálicos , Apoptosis , Muerte Celular , Humanos , Radiación Ionizante , Superóxido DismutasaRESUMEN
The Xeroderma Pigmentosum group C (XPC) protein is indispensable to global genomic repair (GGR), a subpathway of nucleotide excision repair (NER), and plays an important role in the initial damage recognition. XPC can be modified by both ubiquitin and SUMO in response to UV irradiation of cells. Here, we show that XPC undergoes degradation upon UV irradiation, and this is independent of protein ubiquitylation. The subunits of DDB-Cul4A E3 ligase differentially regulate UV-induced XPC degradation, e.g DDB2 is required and promotes, whereas DDB1 and Cul4A protect the protein degradation. Mutation of XPC K655 to alanine abolishes both UV-induced XPC modification and degradation. XPC degradation is necessary for recruiting XPG and efficient NER. The overall results provide crucial insights regarding the fate and role of XPC protein in the initiation of excision repair.
Asunto(s)
Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Animales , Línea Celular , Cricetinae , Proteínas Cullin/fisiología , Daño del ADN , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Endonucleasas/metabolismo , Humanos , Ratones , Proteínas Nucleares/metabolismo , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina/metabolismo , Rayos UltravioletaRESUMEN
The replication checkpoint protein Claspin is important for maintenance of genomic stability and is required for cells to overcome genotoxic stress. Upon UV-induced DNA damage, Claspin is required for activation of the ATR-mediated DNA damage checkpoint response, leading to arrest of DNA replication and inhibition of cell cycle progression. Located at the DNA replication fork, Claspin is also suggested to monitor replication and sense damage. Our present studies in HeLa cells demonstrate associations between the Claspin/ATR-related DNA damage checkpoint response and the global genomic nucleotide excision repair pathway. siRNA-mediated knockdown of Claspin abolishes the UV-induced degradation of DDB2 and impairs the co-localization of DDB2 to DNA damage sites. Thus, the presence of Claspin is required for the total turnover of DNA damage binding protein DDB2, as well as for its functionality in DNA damage recognition. Claspin, however, seems not to be required for maintaining the cellular level of the NER factor XPC and its UV-induced post-translational modifications. Co-localization of XPC with DNA lesions is also intact in the absence of Claspin as is the repair of the UV-induced lesions CPD and 6-4PP. Claspin itself may be directly responsible for physical interaction between the two pathways since Claspin is able to associate with DDB1, DDB2 and XPC. Taken together, these findings reveal physical and functional interplay between Claspin and NER-related proteins and demonstrate crosstalk between the DNA damage checkpoint control and DNA damage repair pathways.