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Cisplatin has been widely used in cancer treatments. Recent evidence indicates that adenine has potential anticancer activities against various types of cancers. However, the effects of the combination of adenine and cisplatin on hepatocellular carcinoma (HCC) cells remain sketchy. Here, our objective was to elucidate the anticancer activity of adenine in combination with cisplatin in HCC cells and its mechanistic pathways. Cell viability and cell cycle progression were assessed by the SRB assay and flow cytometry, respectively. Apoptosis was demonstrated by PI/annexin V staining and flow cytometric analysis. Protein expression, signaling cascade, and mRNA expression were analyzed by Western blotting and quantitative RT-PCR, respectively. Our results showed that adenine jointly potentiated the inhibitory effects of cisplatin on the cell viability of SK-Hep1 and Huh7 cells. Further investigation showed that adenine combined with cisplatin induced higher S phase arrest and apoptosis in HCC cells. Mechanically, adenine induced AMPK activation, reduced mTOR phosphorylation, and increased p53 and p21 levels. The combination of adenine and cisplatin synergistically reduced Bcl-2 and increased PUMA, cleaved caspase-3, and PARP in HCC cells. Adenine also upregulated the mRNA expression of p53, p21, PUMA, and PARP, while knockdown of AMPK reduced the increased expression of these genes. Furthermore, adenine also induced the activation of p38 MAPK through AMPK signaling, and the inhibition of p38 MAPK reduced the apoptosis of HCC cells with exposure to adenine combined with cisplatin. Collectively, these findings reveal that the combination of adenine and cisplatin synergistically enhances apoptosis of HCC cells, which may be attributed to the AMPK-mediated p53/p21 and p38 MAPK cascades. It suggests that adenine may be a potential adjuvant for the treatment of HCC in combination with cisplatin.
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Background: Diabetic foot and leg ulcers are a major cause of disability among patients with diabetes mellitus. A topical gel called ENERGI-F703, applied twice daily and with adenine as its active pharmaceutical ingredient, accelerated wound healing in diabetic mice. The current study evaluated the safety and efficacy of ENERGI-F703 for patients with diabetic foot and leg ulcers. Methods: This randomized, double-blind, multicenter, phase II trial recruited patients from eight medical centers in Taiwan. Patients with intractable diabetic foot and leg ulcers (Wagner Grade 1-3 without active osteomyelitis) were randomly assigned (2:1) to receive topical ENERGI-F703 gel or vehicle gel twice daily for 12 weeks or until complete ulcer closure. The investigator, enrolled patients and site personnel were masked to treatment allocation. Intention to treat (ITT) population and safety population were patient to primary analyses and safety analyses, respectively. Primary outcome was complete ulcer closure rate at the end of treatment. This trial is registered with ClinicalTrials.gov, number NCT02672436. Findings: Starting from March 15th, 2017 to December 26th, 2019, 141 patients were enrolled as safety population and randomized into ENERGI-F703 gel (n = 95) group or vehicle gel (n = 46) group. In ITT population, ENERGI-F703 (n = 90) and vehicle group showed ulcer closure rates of 36.7% (95% CI = 26.75% - 47.49%) and 26.2% (95% CI = 13.86% - 42.04%) with difference of 9.74 % (95 % CI = -6.74% - 26.23%) and 25% quartiles of the time to complete ulcer closure of 69 days and 84 days, respectively. There were 25 (26.3%) patients in ENERGI-F703 group and 11 (23.9%) patients in vehicle group experiencing serious adverse events and five deaths occurred during the study period, none of them related to the treatment. Interpretation: Our study suggests that ENERGI-F703 gel is a safe and well-tolerated treatment for chronic diabetic foot and leg ulcers. Further studies are needed to corroborate our findings in light of limitations. Funding: Energenesis Biomedical Co., Ltd.
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Rosmarinic acid (RA) is a phenolic compound that has several bioactivities, such as anti-inflammatory and antioxidant activities. Here, we further investigate the anti-inflammatory effect of RA on rat A7r5 aortic smooth muscle cells with exposure to lipopolysaccharide (LPS). Our findings showed that low-dose RA (10-25 µM) did not influence the cell viability and morphology of A7r5 cells and significantly inhibited LPS-induced mRNA expression of the pro-inflammatory mediators TNFα, IL-8, and inducible NO synthase (iNOS). Consistently, RA reduced the production of TNFα, IL-8, and NO by A7r5 cells with exposure to LPS. Signaling cascade analysis showed that LPS induced activation of Erk, JNK, p38 mitogen-activated protein kinase (MAPK), and NF-κB, and RA treatments attenuated the activation of the three MAPKs and NF-κB. Moreover, cotreatment with RA and Erk, JNK, p38 MAPK, or NF-κB inhibitors further downregulated the mRNA expression of TNFα, IL-8, and iNOS, and decreased the production of TNFα, IL-8, and NO by A7r5 cells. Taken together, these findings indicate that RA may ameliorate the LPS-provoked inflammatory response of vascular smooth muscle cells by inhibition of MAPK/NF-κB signaling.
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Ischemia followed by blood supply reperfusion in cardiomyocytes leads to an overproduction of free radicals and a rapid decrease of adenosine triphosphate concentration. The cardioprotective effect of a potential drug, adenine, was evaluated using H9c2 rat cardiomyoblasts. After hypoxia-reoxygenation (HR) treatment consisting of hypoxia for 21 h followed by reoxygenation for 6 h, it was revealed that pretreatment with 200 µM adenine for 2 h effectively prevented HR-induced cell death. Adenine also significantly decreased the production of reactive oxygen species and reduced cell apoptosis after HR injury. The antioxidant effect of adenine was also revealed in this study. Adenine pretreatment significantly reduced the expression of activating transcription factor 4 (ATF4) and glucose-regulated protein 78 (GRP78) proteins, and protein disulfide isomerase induced a protective effect on mitochondria after HR stimulation. Intracellular adenosine monophosphate-activated protein kinase, peroxisome proliferator-activated receptor delta (PPARδ), and perilipin levels were increased by adenine after HR stimulation. Adenine had a protective effect in HR-damaged H9c2 cells. It may be used in multiple preventive medicines in the future.
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Tumor metastasis is a major cause of death of patients with colorectal cancer (CRC). Our previous findings show that adenine has antiproliferation activity against tumor cells. However, whether adenine reduces the invasiveness of DLD-1 and SW480 CRC cells has not been thoroughly explored. In this study, we aimed to explore the effects of adenine on the invasion potential of DLD-1 cells. Our findings showed that adenine at concentrations of ≤200 µM did not influence the cell viability of DLD-1 and SW480 CRC cells. By contrast, adenine reduced the migratory potential of the CRC cells. Moreover, it decreased the invasion capacity of the CRC cells in a dose-dependent manner. We further observed that adenine downregulated the protein levels of tissue plasminogen activator, matrix metalloproteinase-9, Snail, TWIST, and vimentin, but upregulated the tissue inhibitor of metalloproteinase-1 expression in DLD-1 cells. Adenine decreased the integrin αV level and reduced the activation of integrin-associated signaling components, including focal adhesion kinase (FAK), paxillin, and Src in DLD-1 cells. Further observations showed that adenine induced AMP-activated protein kinase (AMPK) activation and inhibited mTOR phosphorylation in DLD-1 cells. The knockdown of AMPK restored the reduced integrin αV level and FAK/paxillin/Src signaling inhibited by adenine in DLD-1 cells. Collectively, these findings reveal that adenine reduces the invasion potential of DLD-1 cells through the AMPK/integrin/FAK axis, suggesting that adenine may have anti-metastatic potential in CRC cells.
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Adenine phosphoribosyltransferase (APRT) is the key enzyme involved in purine salvage by the incorporation of adenine and phosphoribosyl pyrophosphate to provide adenylate nucleotides. To evaluate the role of APRT in the repair processes of cutaneous wounds in healthy skin and in diabetic patients, a diabetic mouse model (db/db) and age-matched wild-type mice were used. Moreover, the topical application of adenine was assessed. In vitro studies, analytical, histological, and immunohistochemical methods were used. Diabetic mice treated with adenine exhibited elevated ATP levels in organismic skin and accelerated wound healing. In vitro studies showed that APRT utilized adenine to rescue cellular ATP levels and proliferation from hydrogen peroxide-induced oxidative damage. HPLC-ESI-MS/MS-based analysis of total adenylate nucleotides in NIH-3T3 fibroblasts demonstrated that adenine addition enlarged the cellular adenylate pool, reduced the adenylate energy charge, and provided additional AMP for the further generation of ATP. These data indicate an upregulation of APRT in skin wounds, highlighting its role during the healing of diabetic wounds through regulation of the nucleotide pool after injury. Furthermore, topical adenine supplementation resulted in an enlargement of the adenylate pool needed for the generation of ATP, an important molecule for wound repair.
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Adenina Fosforribosiltransferasa/fisiología , Diabetes Mellitus Experimental/fisiopatología , Cicatrización de Heridas/fisiología , Adenina/farmacología , Adenosina Trifosfato/metabolismo , Animales , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Metabolismo Energético/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Células 3T3 NIH , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Background: Adenine exhibits potential anticancer activity against several types of malignancies. However, whether adenine has anticancer effects on hepatocellular carcinoma (HCC) cells is incompletely explored. Methods: Human HCC cell lines HepG2 and SK-Hep-1 (p53-wild type) and Hep3B (p53-deficient) were used as cell model. Cell growth and cell cycle distribution were determined using MTT assay and flow cytometric analysis, respectively. Protein expression and phosphorylation were assessed by Western blot. Involvement of AMP-activated protein kinase (AMPK) was evaluated using specific inhibitor and small inhibitory RNA (siRNA). Results: Adenine treatments (0.5 - 2 mM) clearly decreased the cell growth of Hep G2 and SK-Hep-1 cells to 72.5 ± 3.4% and 71.3 ± 4.6% of control, respectively. In parallel, adenine also induced sub-G1 and S phase accumulation in both HCC cells. However, adenine did not affect the cell growth and cell cycle distribution of Hep3B cell. Western blot analysis showed that adenine reduced expression of cyclin A/D1 and cyclin-dependent kinase (CDK)2 and upregulated p53, p21, Bax, PUMA, and NOXA in HepG2 cell. Moreover, adenine induced AMPK activation that was involved in the p53-associated apoptotic cascade in HepG2 cells. Inhibition of AMPK activation or knockdown of AMPK restored the decreased cell growth of HepG2 and SK-Hep-1 cells in response to adenine. Conclusions: These findings reveal that adenine reduces the cell growth of HepG2 and SK-Hep-1 but not Hep3B cells, attributing to the AMPK/p53-mediated S phase arrest and apoptosis. It suggests that adenine has anticancer potential against p53-wild type HCC cells and may be beneficial as an adjuvant for HCC treatment.
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Adenina/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Ciclo Celular/efectos de los fármacos , Neoplasias Hepáticas/tratamiento farmacológico , Proteínas Quinasas Activadas por AMP/metabolismo , Adenina/uso terapéutico , Apoptosis/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Células Hep G2 , HumanosRESUMEN
Pathogenic bacteria induced sepsis is a risk factor for hospital mortality. Monocyte-derived inflammatory cytokines participate in the sepsis progression. The anti-inflammatory effect of adenine has been previously reported by our laboratory and others. However, the mechanism of action has different opinions and remains unclear in monocyte. Here, adenine was found to significantly inhibit the secretion of lipopolysaccharide-induced inflammatory cytokines such as TNF-α, IL-1ß and IL-6 in THP-1 cells. The bioinformatic analysis results showed that the anti-inflammatory function is possibly due to the inhibition of NF-κB signaling. And this result is confirmed by using immunocytochemistry. Moreover, this effect can be suppressed by the AMPK inhibitor. Results also showed that adenine can activate AMPK and its multiple downstream targets. Data from mass spectrometry showed that adenine promotes significant elevation of intracellular AMP. Our data indicate that the anti-inflammatory mechanism of adenine may involve adenine phosphoribosyltransferase-catalyzed intracellular AMP elevation, which stimulates AMPK activation.
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Proteínas Quinasas Activadas por AMP/metabolismo , Adenina/farmacología , Antiinflamatorios/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Citocinas/antagonistas & inhibidores , Citocinas/metabolismo , Activación Enzimática , Humanos , Inflamación/inducido químicamente , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/metabolismo , Lipopolisacáridos/farmacología , FN-kappa B/metabolismo , Transducción de Señal , Células THP-1RESUMEN
INTRODUCTION: Numerous studies have investigated the relationship between deregulated HOXB7 expression with the clinical outcome in patients with digestive stem cancers, HOXB7 has showed negative impacts but with varying levels. We aimed to comprehensively evaluate the prediction and prognostic value of HOXB7 in digestive stem cancers. EVIDENCE ACQUISITION: Electronic databases updated to December 1st, 2016 were retrieved to collect relevant eligible studies to quantitatively explore the potential roles of HOXB7 as a prognostic indicator in digestive system cancers. EVIDENCE SYNTHESIS: A total of 9 studies (N.=1298) was included in this synthetical meta-analysis. The pooled hazard ratios suggested that high expression of HOXB7 protein was associated with poor prognosis of OS in patients with digestive system cancers (HR=1.97, 95% CI: 1.65-2.28, P=0.000), and HOXB7 protein could act as an independent prognostic factor for predicting OS of patients with digestive system cancers (HR=2.02, 95% CI: 1.69-2.36, P=0.000). Statistical significance was also observed in subgroup meta-analysis based on the cancer type, histology type, country, sample size and publication date. Furthermore, we examined the correlations between HOXB7 protein and clinicopathological features. It showed that altered expression of HOXB7 protein was correlated with tumor invasion (P=0.000), lymph node status (P=0.000), distant metastasis (P=0.001) and TNM stage (P=0.000). However, the expression of HOXB7 protein was not associated with age (P=0.64), gender (P=0.40) or levels of differentiation (P=0.19). CONCLUSIONS: High expression of HOXB7 protein was associated with poor prognosis of patients with digestive system cancers, as well as clinicopathologic characteristics, including the tumor invasion, lymph node status, distant metastasis and TNM stage. The expression of HOXB7 protein was not associated with age, gender or levels of differentiation. HOXB7 protein expression level in tumor tissue might serve as a novel prognostic marker for digestive system cancers.
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Neoplasias del Sistema Digestivo/genética , Proteínas de Homeodominio/genética , Regulación Neoplásica de la Expresión Génica , Humanos , PronósticoRESUMEN
Proteome analysis of serum from type 2 diabetics with complications may lead to the discovery of diagnostic or prognostic biomarkers. To circumvent the principal barrier of serum proteomics, our investigation aimed to evaluate whether a study of post-translational modification enriched serum proteins could be valuable for the discovery of biomarkers or metabolic pathways related to type 2 diabetes pathogenesis. Type 2 diabetes was induced from high-fat diet fed Sprague Dawley rats with streptozotocin injection. Once diabetic status was confirmed, serum samples from either fasted healthy or diabetic rats were pooled and profiled by two-dimensional difference gel electrophoresis or comparative 2D electrophoresis after protein enrichments using immobilized metal ion, concanavalin A, and lentil affinity chromatography, respectively. Differential expressed proteins were identified and the associated networks were established by an Ingenuity Pathway Analysis. As a result, induced rats became severe diabetic and accompanied by hyperlipidemia, fatty liver, and glomerular hypertrophy. There were 3 total, 14 phosphorylated and 23 glycosylated protein targets differentially expressed. Proteins could be linked to HNF4A, HNF1A, and NFκB transcriptional factors and antigen presentation, humoral immune response, and inflammatory response pathways. Predicted organ toxicity in kidney, heart, and liver matched with our histopathological results. In conclusion, post-translational modification based serum protein enrichment could be a valuable approach to enhance the resolution of serum proteomics without depleting potentially valuable abundant proteins. Our results also indicated the potential association of the hepatic secretome and hepatocyte nuclear factors in the pathogenesis of type 2 diabetes and its complications.
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Proteínas Sanguíneas/metabolismo , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/metabolismo , Procesamiento Proteico-Postraduccional , Proteómica , Animales , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Tipo 2/sangre , Masculino , Ratas , Ratas Sprague-Dawley , Biología de SistemasRESUMEN
Imidazole-zinc reverse stain (ZN stain) is known for high sensitivity, ease of use, and cost-effective feature. ZN stain is compatible to many experiments of which those are proteomics-related in particular. Here, we describe the ZN staining procedures and the subsequent procedures incorporated in detail, along with the improvements of setup in aspects of visualization and documentation for postprocessing ZN stained gel images.
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Electroforesis en Gel de Poliacrilamida , Imidazoles , Proteínas , Coloración y Etiquetado , Zinc , Línea Celular , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Imidazoles/química , Espectrometría de Masas , Proteínas/análisis , Proteínas/química , Coloración y Etiquetado/métodos , Zinc/químicaRESUMEN
Wound healing is one of the major complications of diabetes, and problems with wound healing in diabetics often lead to amputation and even death. AMP-activated protein kinase (AMPK) is a protein involved in intracellular metabolism. Activated AMPK can reduce visceral fat and cholesterol synthesis and even inhibit hepatic gluconeogenesis. Activation of AMPK has been widely used in the treatment of type II diabetes. We applied an AMPK activator (Adenine) to human fibroblasts and to the wounds of streptozotocin-induced diabetic mice. We applied Adenine ointment to the wounds on 7 consecutive days and observed the healing status as well as activation of AMPK and angiogenic factors. Based on the appearance of the wounds, the results showed that after 7 days of treatment the wound area was smaller in the Adenine-treated group relative to the control group. The results for tissue protein expression showed that, compared to the control group, angiogenic related protein, PPARδ were increased and receptor for advanced glycation endproducts (RAGE) was decreased in the Adenine-treated group. Our studies indicate that Adenine has the potential to become a useful drug in the treatment of diabetic wound healing.
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Adenina/farmacología , Diabetes Mellitus Experimental/fisiopatología , Neovascularización Fisiológica/efectos de los fármacos , PPAR delta/metabolismo , Cicatrización de Heridas/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Línea Celular , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Activación Enzimática/efectos de los fármacos , Humanos , Masculino , Ratones , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
AMP-activated protein kinase (AMPK) is known as a pivotal regulator of cellular metabolism. Mounting evidences have demonstrated that AMPK activation exerts tumor suppressive activity on leukemia cells. The present study reported that adenine, an AMPK activator, triggers cell cycle arrest and autophagy of human chronic myelogenous leukemia K562 cells consequently suppressing cell viability. The present findings revealed that adenine treatment (4.0-8.0 mM) significantly inhibited the viability of K562 cells to 69.3±2.5% (24 h) and 53.4±2.1% (48 h) of the control. Flow cytometric analysis revealed that there was a significant accumulation in G2/M phase, but not sub-G1 phase K562 cells following exposure to adenine. Additional investigation demonstrated that adenine treatments significantly increased the number of acidic vesicular organelles and the level of autophagosomal microtubule associated protein 1 light chain 3 α (LC3) marker. By contrast, cleavage of caspase-9, caspase-3 and poly-ADP-ribose polymerase was insignificantly affected in K562 cells following adenine treatment. In K562 cells, adenine was able to markedly promote the phosphorylation of AMPKα and suppress the phosphorylation of mammalian target of rapamycin (mTOR), a downstream target of AMPK. In addition, inhibiting AMPK phosphorylation using dorsomorphin restored mTOR phosphorylation, inhibited the accumulation of LC3 and significantly recovered the suppressed cell viability in response to adenine. Taken together, the present results demonstrated that adenine induced G2/M phase arrest and autophagic cell death, consequently suppressing the viability of K562 cells, which may attribute to the AMPK activation triggered by adenine. These findings provide evidence that adenine may be beneficial to chronic myelogenous leukemia therapy by suppressing excessive cell proliferation.
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Proteomic approaches were applied to investigate whether Photobacterium damselae subsp. piscicida (Phdp) can directly sense and respond to growth conditions under different salinities, 0.85% and 3.5% NaCl concentrations, mimicking the osmotic conditions in host and marine water bodies, respectively. Proteins significantly altered were analyzed by two-dimensional gel electrophoresis (2-DE), liquid chromatography-electrospray ionization-quadrupole-time-of-flight tandem mass spectrometry (LC-ESI-Q-TOF MS/MS) and bioinformatics analysis, thus resulting in 16 outer membrane proteins (OMPs), 12 inner membrane proteins (IMPs), and 20 cytoplasmic proteins (CPs). Quantitative real-time PCR was also applied to monitor the mRNA expression level of these target proteins. Cluster of orthologous groups of protein (COG) analysis revealed that when shifting from 3.5% to 0.85% salinity, the majority of the up-regulated proteins were involved in posttranslational modification, protein turnover, and chaperones, while the down-regulated proteins were mainly related to energy production and conversion, compatible solutes (carbohydrates, amino acids and their derivatives) biogenesis and transport. Differentially expressed proteins identified in the current study could be used to elucidate the salt adaptation mechanisms of Phdp during their transition between host cells and the marine habitats.
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Photobacterium/genética , Photobacterium/fisiología , Proteoma , Tolerancia a la Sal , Animales , Electroforesis en Gel Bidimensional , Enfermedades de los Peces/microbiología , Osmorregulación/genética , Photobacterium/crecimiento & desarrollo , Photobacterium/ultraestructura , Proteómica , Reacción en Cadena en Tiempo Real de la Polimerasa , Tolerancia a la Sal/genética , Cloruro de Sodio/farmacología , Espectrometría de Masas en TándemRESUMEN
Disulfide linkages play an important role in protein stability and activity. Thus, it is critical to characterize disulfide bonds to ensure the quality and function of protein pharmaceuticals. There are, however, problems associated with maintaining disulfide linkages in the conventional procedures that are used to digest a protein. In order to preserve enzyme activity during the digestion of a protein, it is commonly carried out at neutral to basic environment which increases the possibilities of disulfide bond scrambling. However, it is not easy to differentiate whether the scrambled disulfide linkages are initiated by the sample itself or whether they are induced during the protease digestion process. In this study, the optimum pH for minimizing disulfide bond rearrangements during the digestion process was determined. Three sets of proteases, trypsin plus Glu-C, Lys-C and thermolysin were used, followed by dimethyl labeling and mass spectrometry for a bevacizumab (Avastin) disulfide linkage analysis. No disulfide linkage scrambling was detected at pH6 when Lys-C or trypsin plus Glu-C were used as enzymes. When thermolysin was applied, some scrambled disulfide bonds were identified at pH5, 6 and 7. Nevertheless, there was less disulfide bond scrambling at a lower pH. All correct disulfide bonds on bevacizumab could be identified using this approach. The results demonstrated that by choosing the proper enzymes, using a lower pH environment for the digestion could reduce the degree of artifact disulfide scrambling.
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Inhibidores de la Angiogénesis/química , Bevacizumab/química , Disulfuros/química , Termolisina/química , Tripsina/química , Secuencia de Aminoácidos , Biocatálisis , Concentración de Iones de Hidrógeno , Hidrólisis , Espectrometría de Masas , SolucionesAsunto(s)
Adenina/farmacología , Senescencia Celular/efectos de los fármacos , Dermis/citología , Folículo Piloso/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , División Celular/efectos de los fármacos , Células Cultivadas , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/efectos de los fármacos , Folículo Piloso/citología , Folículo Piloso/enzimología , Humanos , NAD/metabolismo , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ribonucleótidos/farmacología , beta-Galactosidasa/análisisRESUMEN
The aim of this study was to investigate the anti-inflammatory effects and mechanism of action of the gold nanoparticles (AuNPs) on vascular injury. In vitro vascular endothelial cell (EC) inflammation and in vivo rat carotid balloon injury models were used. The expression of TNF-α-induced cell adhesion molecules (CAMs) was suppressed by the AuNPs in human umbilical vein ECs and aortic ECs. The AuNPs reduced TNF-α-induced intracellular ROS production and NF-κB signaling pathways and enhanced CAM protein degradation by increasing their ubiquitination. However, they did not interfere with the mTOR pathway for protein synthesis and TNF-αbinding to ECs. These effects led to a reduction of monocyte adhesion to EC monolayers in vitro and endothelial CAM expression and monocyte/macrophage level in the vascular injured areas, contributing to a substantial decrease of arterial neointima formation in the rat carotid balloon injury model. The serum gold concentration was 99.5±18 ng/ml after three-day oral administration. Moreover, incubation of the AuNPs with serum and albumin led to an increase of particle sizes of the AuNPs. Collectively, we provide the first evidence that demonstrates that AuNPs possess anti-inflammatory bioactivity on vascular ECsin vitro and can reduce arterial neointima hyperplasia during vascular injury in vivo.
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Moléculas de Adhesión Celular/metabolismo , Oro/farmacología , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Nanopartículas del Metal/química , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Angioplastia de Balón/efectos adversos , Animales , Modelos Animales de Enfermedad , Oro/química , Humanos , Neointima/metabolismo , RatasRESUMEN
The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.
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Proteínas Quinasas Activadas por AMP/metabolismo , Adenina/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Adenina/toxicidad , Adenina Fosforribosiltransferasa/genética , Adenina Fosforribosiltransferasa/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacología , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Monocitos/metabolismo , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Ribonucleótidos/farmacologíaRESUMEN
Malignant gliomas, which comprise the most common type of primary malignant brain tumor, are associated with a poor prognosis and quality of life. Paclitaxel (Taxol) and temozolomide (TMZ) are Food and Drug Administrationapproved anticancer agents, which are known to have therapeutic applications in various malignancies. However, similar to other chemotherapeutic agents, the development of resistance to TMZ and Taxol is common. The aim of the present study was to investigate the regulation of glucose metabolism by TMZ and Taxol in glioma cells. The results demonstrated that glioma cells exhibit decreased glucose uptake and lactate production in response to treatment with TMZ; however, glucose metabolism was increased in response to Taxol treatment. Following analysis of TMZ and Taxolresistant cell lines, it was reported that glucose metabolism was decreased in the TMZresistant cells, but was increased in the Taxolresistant cells. Notably, a combination of TMZ and Taxol exerted synergistic inhibitory effects on Taxolresistant glioma cells. However, the synergistic phenotype was not observed following treatment with a combination of 5fluorouracil and Taxol. Furthermore, restoration of glucose metabolism by overexpression of glucose transporter 1 in Taxolresistant cells resulted in regained resistance to Taxol. Therefore, the present study proposes a novel mechanism accounting for the synergistic effects of Taxol and TMZ cotreatment, which may contribute to the development of therapeutic strategies for overcoming chemoresistance in patients with cancer.
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Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Dacarbazina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Glioma/tratamiento farmacológico , Glucosa/metabolismo , Paclitaxel/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Dacarbazina/farmacología , Sinergismo Farmacológico , Glioma/metabolismo , Glioma/patología , Humanos , TemozolomidaRESUMEN
The precipitation of monosodium urate crystals within joints triggers an acute inflammatory reaction that is the root cause of gout. The inflammation induced by the injection of MSU crystals into the murine air pouch for 1, 3, and 5 h was examined by iTRAQ-based proteomic profiling. The iTRAQ-labeled peptides were fractionated by SCX, basic-RP or solution-IEF, followed by LC-MS/MS analysis. A total of 951 proteins were quantified from the total combined fractions. Among them, 317 proteins exhibited a differential expression, compared to that of the controls at one time point or more. The majority of the differentially expressed proteins were found in the sample after a 5-h MSU treatment. Western blot revealed that the expression levels of cathelin-related antimicrobial peptide and S100A9 were positively correlated with the time-course treated with MSU. Further analysis of GeneGO pathway demonstrated that these differentially expressed proteins are primarily related to the immune-related complement system and the tricarboxylic acid cycle. Moreover, seven genes from the TCA cycle were found to be significantly downregulated at the transcriptional level and its correlation with gout and possible therapeutic applications are worth further investigation. Last, we found that pyruvate carboxylation could be potential targets for antigout treatment.