RESUMEN
Organometallic metal(arene) anticancer agents require ligand exchange for their anticancer activity and this is generally believed to confer low selectivity for potential cellular targets. However, using an integrated proteomics-based target-response profiling approach as a potent hypothesis-generating procedure, we found an unexpected target selectivity of a ruthenium(arene) pyridinecarbothioamide (plecstatin) for plectin, a scaffold protein and cytolinker, which was validated in a plectin knock-out model inâ vitro. Plectin targeting shows potential as a strategy to inhibit tumor invasiveness as shown in cultured tumor spheroids while oral administration of plecstatin-1 to mice reduces tumor growth more efficiently in the invasive B16â melanoma than in the CT26â colon tumor model.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Organometálicos/farmacología , Plectina/efectos de los fármacos , Compuestos de Rutenio/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Técnicas de Inactivación de Genes , Ontología de Genes , Humanos , Ratones , Neoplasias Experimentales/patología , Compuestos Organometálicos/química , Plectina/genética , Compuestos de Rutenio/químicaRESUMEN
Metalation of hen egg white lysozyme (HEWL) with organometallics was studied with physicochemical methods in solid state, solution and the gas phase. While metalation did not affect the crystal structure of HEWL significantly, protein destabilisation was detected in gas phase and solution.
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Immune-evasion and immune checkpoints are promising new therapeutic targets for several cancer entities. In ovarian cancer, the clinical role of programmed cell death receptor ligand 1 (PD-L1) expression as mechanism to escape immune recognition has not been clarified yet. We analyzed PD-L1 expression of primary ovarian and peritoneal tumor tissues together with several other parameters (whole transcriptomes of isolated tumor cells, local and systemic immune cells, systemic cytokines and metabolites) and compared PD-L1 expression between primary tumor and tumor recurrences. All expressed major histocompatibility complex (MHC) I genes were negatively correlated to PD-L1 abundances on tumor tissues, indicating two mutually exclusive immune-evasion mechanisms in ovarian cancer: either down-regulation of T-cell mediated immunity by PD-L1 expression or silencing of self-antigen presentation by down-regulation of the MHC I complex. In our cohort and in most of published evidences in ovarian cancer, low PD-L1 expression is associated with unfavorable outcome. Differences in immune cell populations, cytokines, and metabolites strengthen this picture and suggest the existence of concurrent pathways for progression of this disease. Furthermore, recurrences showed significantly increased PD-L1 expression compared to the primary tumors, supporting trials of checkpoint inhibition in the recurrent setting.
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Antígenos de Histocompatibilidad Clase I/genética , Neoplasias Ováricas/patología , Receptor de Muerte Celular Programada 1/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Quimiocinas/metabolismo , Citocinas/metabolismo , Femenino , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Estimación de Kaplan-Meier , Células Asesinas Naturales/citología , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Neoplasias Ováricas/mortalidad , Receptor ErbB-2/sangreRESUMEN
During inflammation, proteins and lipids act in a concerted fashion, calling for combined analyses. Fibroblasts are powerful mediators of chronic inflammation. However, little is known about eicosanoid formation by human fibroblasts. The aim of this study was to analyze the formation of the most relevant inflammation mediators including proteins and lipids in human fibroblasts upon inflammatory stimulation and subsequent treatment with dexamethasone, a powerful antiphlogistic drug. Label-free quantification was applied for proteome profiling, while an in-house established data-dependent analysis method based on high-resolution mass spectrometry was applied for eicosadomics. Furthermore, a set of 188 metabolites was determined by targeted analysis. The secretion of 40 proteins including cytokines, proteases, and other inflammation agonists as well as 14 proinflammatory and nine anti-inflammatory eicosanoids was found significantly induced, while several acylcarnithins and sphingomyelins were found significantly downregulated upon inflammatory stimulation. Treatment with dexamethasone downregulated most cytokines and proteases, abrogated the formation of pro- but also anti-inflammatory eicosanoids, and restored normal levels of acylcarnithins but not of sphingomyelins. In addition, the chemokines CXCL1, CXCL5, CXCL6, and complement C3, known to contribute to chronic inflammation, were not counter-regulated by dexamethasone. Similar findings were obtained with human mesenchymal stem cells, and results were confirmed by targeted analysis with multiple reaction monitoring. Comparative proteome profiling regarding other cells demonstrated cell-type-specific synthesis of, among others, eicosanoid-forming enzymes as well as relevant transcription factors, allowing us to better understand cell-type-specific regulation of inflammation mediators and shedding new light on the role of fibroblasts in chronic inflammation.
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Eicosanoides/metabolismo , Fibroblastos/metabolismo , Inflamación/metabolismo , Metabolómica , Proteoma , Antiinflamatorios/farmacología , Células Cultivadas , Quimiocinas/metabolismo , Cromatografía Liquida/métodos , Enfermedad Crónica , Citocinas/metabolismo , Dexametasona/farmacología , Fibroblastos/efectos de los fármacos , Humanos , Inflamación/sangre , Inflamación/patología , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/metabolismo , Espectrometría de Masas/métodosRESUMEN
Response profiling using shotgun proteomics for establishing global metallodrug mechanisms of action in two colon carcinoma cell lines, HCT116 and SW480, has been applied and evaluated with the clinically approved arsenic trioxide. Surprisingly, the complete established mechanism of action of arsenic trioxide was observed by protein regulations in SW480, but not HCT116 cells. Comparing the basal protein expression in the two cell lines revealed an 80 % convergence of protein identification, but with significant expression differences, which in turn seem to affect the extent of protein regulation. A clear-cut redox response was observed in SW480 cells upon treatment with arsenic, but hardly in HCT116 cells. Response profiling was then used to investigate four anti-cancer metallodrugs (KP46, KP772, KP1339 and KP1537). Proteome alterations were mapped to selected functional groups, including DNA repair, endocytosis, protection from oxidative stress, protection from endoplasmatic reticulum (ER) stress, cell adhesion and mitochondrial function. The present data suggest that knowledge of the mechanism of action of anti-cancer metallodrugs and improved patient stratification strategies are imperative for the design of clinical studies.
RESUMEN
Peripheral blood mononuclear cells are important players in immune regulation relying on a complex network of signaling pathways. In this study, we evaluated the power of label-free quantitative shotgun proteomics regarding the comprehensive characterization of signaling pathways in such primary cells by studying regulation of protein abundance, post-translational modifications and nuclear translocation events. The effects of inflammatory stimulation and the treatment of stimulated cells with dexamethasone were investigated. Therefore, a previously published dataset accessible via ProteomeXchange consisting of 6901 identified protein groups was re-evaluated. These data enabled us to comprehensively map the c-JUN, ERK5 and NF-κB signaling cascade in a semi-quantitative fashion. Without the application of any enrichment, 3775 highly confident phosphopeptides derived from 1249 proteins including 66 kinases were identified. Efficient subcellular fractionation and subsequent comparative analysis identified previously unrecognized inflammation-associated nuclear translocation events of proteins such as histone-modifying proteins, zinc finger proteins as well as transcription factors. Profound effects of inflammatory stimulation and dexamethasone treatment on histone H3 and ZFP161 localization represent novel findings and were verified by immunofluorescence. In conclusion, we demonstrate that multiple regulatory events resulting from the activity of signaling pathways can be determined out of untargeted shotgun proteomics data. SIGNIFICANCE: Relevant functional events such as phosphorylation and nuclear translocation of proteins were extracted from high-resolution mass spectrometry data and provided additional biological information contained in shotgun proteomics data.
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Transporte Activo de Núcleo Celular/efectos de los fármacos , Inflamación , Leucocitos Mononucleares/metabolismo , Proteínas/análisis , Proteómica/métodos , Transducción de Señal , Animales , Células Cultivadas , Dexametasona/farmacología , Histonas/metabolismo , Humanos , Inflamación/inducido químicamente , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Leucocitos Mononucleares/química , Espectrometría de Masas , Proteína Quinasa 7 Activada por Mitógenos/metabolismo , FN-kappa B/metabolismo , Fosfopéptidos , Fosforilación , Transporte de Proteínas , Proteínas/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal/efectos de los fármacosRESUMEN
Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia.
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Biomarcadores de Tumor/sangre , Caquexia/metabolismo , Calcio/metabolismo , Melanoma/complicaciones , Metabolómica/métodos , Proteómica/métodos , Asparagina/metabolismo , Plaquetas/metabolismo , Glicina/metabolismo , Humanos , Melanoma/metabolismo , Metástasis de la Neoplasia , Estrés Oxidativo , Fosfatidilcolinas/metabolismo , Activación PlaquetariaRESUMEN
Purpose: Cancer metabolism is characterized by alterations including aerobic glycolysis, oxidative phosphorylation, and need of fuels and building blocks.Experimental Design: Targeted metabolomics of preoperative and follow-up sera, ascites, and tumor tissues, RNA sequencing of isolated tumor cells, local and systemic chemokine, and local immune cell infiltration data from up to 65 high-grade serous ovarian cancer patients and 62 healthy controls were correlated to overall survival and integrated in a Systems Medicine manner.Results: Forty-three mainly (poly)unsaturated glycerophospholipids and four essential amino acids (citrulline) were significantly reduced in patients with short compared with long survival and healthy controls. The glycerophospholipid fingerprint is identical to the fingerprint from isolated (very) low-density lipoproteins (vLDL), indicating that the source of glycerophospholipids consumed by tumors is (v)LDL. A glycerophospholipid-score (HR, 0.46; P = 0.007) and a 100-gene signature (HR, 0.65; P = 0.004) confirmed the independent impact on survival in training (n = 65) and validation (n = 165) cohorts. High concentrations of LDLs and glycerophospholipids were independently predictors for favorable survival. Patients with low glycerophospholipids presented with more systemic inflammation (C-reactive protein and fibrinogen negatively and albumin positively correlated) but less adaptive immune cell tumor infiltration (lower tumor and immune cell PD-L1 expression), less oxygenic respiration and increased triglyceride biosynthesis in tumor cells, and lower histone expressions, correlating with higher numbers of expressed genes and more transcriptional noise, a putative neo-pluripotent tumor cell phenotype.Conclusions: Low serum phospholipids and essential amino acids are correlated with worse outcome in ovarian cancer, accompanied by a specific tumor cell phenotype. Clin Cancer Res; 23(8); 2081-92. ©2016 AACR.
Asunto(s)
Biomarcadores de Tumor/análisis , Cistadenocarcinoma Seroso/metabolismo , Metabolómica/métodos , Neoplasias Ováricas/metabolismo , Anciano , Cistadenocarcinoma Seroso/diagnóstico , Cistadenocarcinoma Seroso/mortalidad , Femenino , Humanos , Estimación de Kaplan-Meier , Persona de Mediana Edad , Clasificación del Tumor , Neoplasias Ováricas/diagnóstico , Neoplasias Ováricas/mortalidad , Pronóstico , Modelos de Riesgos Proporcionales , TranscriptomaRESUMEN
SCOPE: A bolus administration of 0.15 mg nonivamide has previously been demonstrated to reduce energy intake in moderately overweight men. This 12-week intervention investigated whether a daily consumption of nonivamide in a protein-based product formulation promotes a reduction in body weight in healthy overweight subjects and affects outcome measures associated with mechanisms regulating food intake, e.g. plasma concentrations of (an)orexigenic hormones, energy substrates as well as changes in fecal microbiota. METHODS AND RESULTS: Nineteen overweight subjects were randomly assigned to either a control (C) or a nonivamide (NV) group. Changes in the body composition and plasma concentrations of satiating hormones were determined at fasting and 15, 30, 60, 90, and 120 min after a glucose load. Participants were instructed to consume 0.15 mg nonivamide per day in 450 mL of a milk shake additionally to their habitual diet. After treatment, a group difference in body fat mass change (-0.61 ± 0.36% in NV and +1.36 ± 0.38% in C) and an increase in postprandial plasma serotonin were demonstrated. Plasma metabolome and fecal microbiome read outs were not affected. CONCLUSIONS: A daily intake of 0.15 mg nonivamide helps to support to maintain a healthy body composition.
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Capsaicina/análogos & derivados , Grasas de la Dieta/efectos adversos , Sobrepeso/tratamiento farmacológico , Serotonina/sangre , Canales Catiónicos TRPV/agonistas , Aumento de Peso/efectos de los fármacos , Adiponectina/sangre , Adiposidad/efectos de los fármacos , Adulto , Glucemia/metabolismo , Composición Corporal , Índice de Masa Corporal , Peso Corporal , Capsaicina/administración & dosificación , Colesterol/sangre , Dieta , Femenino , Hormonas Gastrointestinales/sangre , Microbioma Gastrointestinal/efectos de los fármacos , Humanos , Insulina/sangre , Leptina/sangre , Masculino , Periodo Posprandial , Saciedad , Fármacos del Sistema Sensorial/administración & dosificación , Triglicéridos/sangre , Adulto JovenRESUMEN
Hydroxypyr(id)ones constitute an emerging platform for the design of drug molecules, owing to their favorable biocompatibility and toxicity profiles. Herein, [RuII (η6 -p-cymene)] complexes with 3-hydroxy-2-pyridinone functionalized with morpholine and thiomorpholine, as a means often used in medicinal chemistry to alter the physicochemical properties of drug compounds, are reported. The compounds underwent hydrolysis of the Ru-Cl bond and the aqua species were stable for up to 48â h in aqueous solution, as observed by 1 Hâ NMR spectroscopy and ESI-MS. The compounds formed adducts with amino acids and proteins through cleavage of the pyridinone ligand. Binding experiments to the nucleosome core particle by means of X-ray crystallography revealed similar reactivity and exclusive binding to histidine moieties of the histone proteins. Preliminary cyclin-dependent kinaseâ 2 (CDK2)/cyclinâ A kinase inhibitory studies revealed promising activity similar to that of structurally related organometallic compounds.
RESUMEN
The immune system plays a critical role in cancer progression and overall survival. Still, it is unclear if differences in the immune response are associated with different patterns of tumor spread apparent in high grade serous ovarian cancer patients and previously described by us. In this study we aimed to assess the role of the immune system in miliary (widespread, millet-sized lesions) and non-miliary (bigger, exophytically growing implants) tumor spread. To achieve this we comprehensively analyzed tumor tissues, blood, and ascites from 41 patients using immunofluorescence, flow cytometry, RNA sequencing, multiplexed immunoassays, and immunohistochemistry. Results showed that inflammation markers were systemically higher in miliary. In contrast, in non-miliary lymphocyte and monocyte/macrophage infiltration into the ascites was higher as well as the levels of PD-1 expression in tumor associated cytotoxic T-lymphocytes and PD-L1 expression in tumor cells. Furthermore, in ascites of miliary patients more epithelial tumor cells were present compared to non-miliary, possibly due to the active down-regulation of anti-tumor responses by B-cells and regulatory T-cells. Summarizing, adaptive immune responses prevailed in patients with non-miliary spread, whereas in patients with miliary spread a higher involvement of the innate immune system was apparent while adaptive responses were counteracted by immune suppressive cells and factors.
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Neoplasias Ováricas/inmunología , Neoplasias Ováricas/metabolismo , Neoplasias Peritoneales/inmunología , Neoplasias Peritoneales/metabolismo , Inmunidad Adaptativa , Antígenos de Neoplasias/inmunología , Ascitis , Linfocitos B/metabolismo , Separación Celular , Progresión de la Enfermedad , Regulación hacia Abajo , Femenino , Citometría de Flujo , Humanos , Inmunidad Innata , Inmunoensayo , Inmunohistoquímica , Inflamación , Macrófagos/metabolismo , Metabolómica , Monocitos/citología , Neoplasias Ováricas/patología , Neoplasias Peritoneales/secundario , Análisis de Secuencia de ARN , Linfocitos T Reguladores/metabolismo , Resultado del Tratamiento , Microambiente TumoralRESUMEN
SCOPE: Anti-inflammatory effects of coffee consumption have been reported to be caused by caffeine and adenosine receptor signaling. However, contradictory effects have been observed. Many kinds of chronic diseases are linked to inflammation; therefore a profound understanding of potential effects of coffee consumption is desirable. METHODS AND RESULTS: We performed ex vivo experiments with eight individuals investigating peripheral blood mononuclear cells isolated from venous blood before and after coffee consumption, as well as in vitro experiments applying caffeine on isolated cells. After in vitro inflammatory stimulation of the cells, released cytokines, chemokines, and eicosanoids were determined and quantified using targeted mass spectrometric methods. Remarkably, the release of inflammation mediators IL6, IL8, GROA, CXCL2, CXCL5 as well as PGA2, PGD2, prostaglandin E2 (PGE2), LTC4, LTE4, and 15S-HETE was significantly affected after coffee consumption. While in several individuals coffee consumption or caffeine treatment caused significant downregulation of most inflammation mediators, in other healthy individuals exactly the opposite effects were observed. CONCLUSION: Ruling out age, sex, coffee consumption habits, the metabolic kinetics of caffeine in blood and the individual amount of regulatory T cells or CD39 expression as predictive parameters, we demonstrated here that coffee consumption may have significant pro- or anti-inflammatory effects in an individual fashion.
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Café/química , Inflamación/sangre , Adulto , Cafeína/administración & dosificación , Células Cultivadas , Quimiocinas/sangre , Quimiocinas/genética , Citocinas/sangre , Citocinas/genética , Eicosanoides/sangre , Eicosanoides/genética , Femenino , Humanos , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
The reactivity of three cytotoxic organometallic gold(III) complexes with cyclometalated C,N,N and C,N ligands (either six- or five-membered metallacycles), as well as that of two representative gold(III) complexes with N-donor ligands, with biological nucleophiles has been studied by ESI-MS on ion trap and time-of-flight instruments. Specifically, the gold compounds were reacted with mixtures of nucleophiles containing l-histidine (imine), l-methionine (thioether), l-cysteine (thiol), l-glutamic acid (carboxylic acid), methylseleno-l-cysteine (selenoether), and in situ generated seleno-l-cysteine (selenol) to judge the preference of the gold compounds for binding to selenium-containing amino acid residues. Moreover, the gold compounds' reactivity was studied with proteins and nucleic acid building blocks. These experiments revealed profound differences between the coordination and organometallic families and even within the family of organometallics, which allowed insights to be gained into the compounds mechanisms of action. In particular, interactions with seleno-l-cysteine appear to reflect well the compounds' inhibition properties of the seleno-enzyme thioredoxin reductase and to a certain extent their antiproliferative effects in vitro. Therefore, mass spectrometry is successfully applied for linking the molecular reactivity and target preferences of metal-based drug candidates to their biological effects. Finally, this experimental setup is applicable to any other metallodrug that undergoes ligand substitution reactions and/or redox changes as part of its mechanism of action.
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Compuestos Orgánicos de Oro/química , Adenosina Trifosfato/química , Animales , Bovinos , Citocromos c/química , Estabilidad de Medicamentos , Guanina/análogos & derivados , Guanina/química , Guanosina Trifosfato/química , Caballos , Selenocisteína/análogos & derivados , Selenocisteína/química , Espectrometría de Masa por Ionización de Electrospray , Tiorredoxina Reductasa 1/química , Ubiquitina/química , Agua/químicaRESUMEN
Organometallics with N-heterocyclic carbene (NHC) ligands have triggered major interest in inorganic medicinal chemistry. Complexes of the type Rh(I)(NHC)(COD)X (where X is Cl or I, COD is cyclooctadiene, and NHC is a dimethylbenzimidazolylidene) represent a promising type of new metallodrugs that have been explored by advanced biomedical methods only recently. In this work, we have synthesized and characterized several complexes of this type. As observed by mass spectrometry, these complexes remained stable over at least 3 h in aqueous solution, after which hydrolysis of the halido ligands occurred and release of the NHC ligand was evident. Effects against mitochondria and general cell tumor metabolism were noted at higher concentrations, whereas phosphorylation of HSP27, p38, ERK1/2, FAK, and p70S6K was induced substantially already at lower exposure levels. Regarding the antiproliferative activity in tumor cells, a clear preference for iodido over chlorido secondary ligands was noted, as well as effects of the substituents of the NHC ligand.
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Antineoplásicos/química , Bencimidazoles/química , Complejos de Coordinación/química , Ciclooctanos/química , Rodio , Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Bencimidazoles/síntesis química , Bencimidazoles/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/farmacología , Ciclooctanos/síntesis química , Ciclooctanos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Estabilidad de Medicamentos , Humanos , Hidrólisis , Mitocondrias/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-ActividadRESUMEN
Classical drug assays are often confined to single molecules and targeting single pathways. However, it is also desirable to investigate the effects of complex mixtures on complex systems such as living cells including the natural multitude of signalling pathways. Evidence based on herbal medicine has motivated us to investigate potential beneficial health effects of Mucor racemosus (M rac) extracts. Secondary metabolites of M rac were collected using a good-manufacturing process (GMP) approved production line and a validated manufacturing process, in order to obtain a stable product termed SyCircue (National Drug Code USA: 10424-102). Toxicological studies confirmed that this product does not contain mycotoxins and is non-genotoxic. Potential effects on inflammatory processes were investigated by treating stimulated cells with M rac extracts and the effects were compared to the standard anti-inflammatory drug dexamethasone on the levels of the proteome and metabolome. Using 2D-PAGE, slight anti-inflammatory effects were observed in primary white blood mononuclear cells, which were more pronounced in primary human umbilical vein endothelial cells (HUVECs). Proteome profiling based on nLC-MS/MS analysis of tryptic digests revealed inhibitory effects of M rac extracts on pro-inflammatory cytoplasmic mediators and secreted cytokines and chemokines in these endothelial cells. This finding was confirmed using targeted proteomics, here treatment of stimulated cells with M rac extracts down-regulated the secretion of IL-6, IL-8, CXCL5 and GROA significantly. Finally, the modulating effects of M rac on HUVECs were also confirmed on the level of the metabolome. Several metabolites displayed significant concentration changes upon treatment of inflammatory activated HUVECs with the M rac extract, including spermine and lysophosphatidylcholine acyl C18:0 and sphingomyelin C26:1, while the bulk of measured metabolites remained unaffected. Interestingly, the effects of M rac treatment on lipids were orthogonal to the effect of dexamethasone underlining differences in the overall mode of action.
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Antiinflamatorios/farmacología , Productos Biológicos/farmacología , Dexametasona/farmacología , Metabolómica/métodos , Mucor/química , Proteómica/métodos , Secuencia de Aminoácidos , Antiinflamatorios/aislamiento & purificación , Productos Biológicos/aislamiento & purificación , Células Cultivadas , Cromatografía Liquida , Citocinas/metabolismo , Electroforesis en Gel Bidimensional , Células Hep G2 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Datos de Secuencia Molecular , Mucor/metabolismo , Pruebas de Mutagenicidad , Proteoma/metabolismo , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Espectrometría de Masas en TándemRESUMEN
A series of heteropentanuclear oxalate-bridged Ru(NO)-Ln (4d-4f) metal complexes of the general formula (nBu4N)5[Ln{RuCl3(µ-ox)(NO)}4], where Ln=Y (2), Gd (3), Tb (4), Dy (5) and ox=oxalate anion, were obtained by treatment of (nBu4N)2[RuCl3(ox)(NO)] (1) with the respective lanthanide salt in 4:1 molar ratio. The compounds were characterized by elemental analysis, IR spectroscopy, electrospray ionization (ESI) mass spectrometry, while 1, 2, and 5 were in addition analyzed by X-ray crystallography, 1 by Ru K-edge XAS and 1 and 2 by (13)Câ NMR spectroscopy. X-ray diffraction showed that in 2 and 5 four complex anions [RuCl3(ox)(NO)](2-) are coordinated to Y(III) and Dy(III), respectively, with formation of [Ln{RuCl3(µ-ox)(NO)}4](5-) (Ln=Y, Dy). While Y(III) is eight-coordinate in 2, Dy(III) is nine-coordinate in 5, with an additional coordination of an EtOH molecule. The negative charge is counterbalanced by five nBu4N(+) ions present in the crystal structure. The stability of complexes 2 and 5 in aqueous medium was monitored by UV/Vis spectroscopy. The antiproliferative activity of ruthenium-lanthanide complexes 2-5 were assayed in two human cancer cell lines (HeLa and A549) and in a noncancerous cell line (MRC-5) and compared with those obtained for the previously reported Os(NO)-Ln (5d-4f) analogues (nBu4N)5[Ln{OsCl3(ox)(NO)}4] (Ln=Y (6), Gd (7), Tb (8), Dy (9)). Complexes 2-5 were found to be slightly more active than 1 in inhibiting the proliferation of HeLa and A549 cells, and significantly more cytotoxic than 5d-4f metal complexes 6-9 in terms of IC50 values. The highest antiproliferative activity with IC50 values of 20.0 and 22.4â µM was found for 4 in HeLa and A549 cell lines, respectively. These cytotoxicity results are in accord with the presented ICP-MS data, indicating five- to eightfold greater accumulation of ruthenium versus osmium in human A549 cancer cells.
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Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Óxidos de Nitrógeno/química , Antineoplásicos/química , Línea Celular Tumoral , Cristalografía por Rayos X , Células HeLa , Humanos , Concentración 50 Inhibidora , Ligandos , Estructura Molecular , Rutenio/químicaRESUMEN
Ruthenium(II)-arene complexes with biotin-containing ligands were prepared so that a novel drug delivery system based on tumor-specific vitamin-receptor mediated endocytosis could be developed. The complexes were characterized by spectroscopic methods and their in vitro anticancer activity in cancer cell lines with various levels of major biotin receptor (COLO205, HCT116 and SW620 cells) was tested in comparison with the ligands. In all cases, coordination of ruthenium resulted in significantly enhanced cytotoxicity. The affinity of Ru(II) -biotin complexes to avidin was investigated and was lower than that of unmodified biotin. Hill coefficients in the range 2.012-2.851 suggest strong positive cooperation between the complexes and avidin. To estimate the likelihood of binding to the biotin receptor/transporter, docking studies with avidin and streptavidin were conducted. These explain, to some extent, the in vitro anticancer activity results and support the conclusion that these novel half-sandwich ruthenium(II)-biotin conjugates may act as biological vectors to cancer cells, although no clear relationship between the cellular Ru content, the cytotoxicity, and the presence of the biotin moiety was observed.
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Biotina/química , Rutenio/química , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Humanos , Estructura MolecularRESUMEN
The clinical development of anticancer metallodrugs is often hindered by the elusive nature of their molecular targets. To identify the molecular targets of an antimetastatic ruthenium organometallic complex based on 1,3,5-triaza-7-phosphaadamantane (RAPTA), we employed a chemical proteomic approach. The approach combines the design of an affinity probe featuring the pharmacophore with mass-spectrometry-based analysis of interacting proteins found in cancer cell lysates. The comparison of data sets obtained for cell lysates from cancer cells before and after treatment with a competitive binder suggests that RAPTA interacts with a number of cancer-related proteins, which may be responsible for the antiangiogenic and antimetastatic activity of RAPTA complexes. Notably, the proteins identified include the cytokines midkine, pleiotrophin and fibroblast growth factor-binding protein 3. We also detected guanine nucleotide-binding protein-like 3 and FAM32A, which is in line with the hypothesis that the antiproliferative activity of RAPTA compounds is due to induction of a G2/M arrest and histone proteins identified earlier as potential targets.
RESUMEN
Chemotherapeutics for the treatment of tumorigenic conditions that feature novel modes of action are highly sought after to overcome the limitations of current chemotherapies. Herein, we report the conjugation of the alkylating agent chlorambucil to the RAPTA scaffold, a well-established pharmacophore. While chlorambucil is known to alkylate DNA, the RAPTA complexes are known to coordinate to amino acid side chains of proteins. Therefore, such a molecule combines DNA and protein targeting properties in a single molecule. Several chlorambucil-tethered RAPTA derivatives were prepared and tested for their cytotoxicity, stability in water and reactivity to protein and DNA substrates. The anticancer activity of the complexes is widely driven by the cytotoxicity of the chlorambucil moiety. However, especially in the cisplatin-resistant A2780R cells, the chlorambucil-functionalized RAPTA derivatives are in general more cytotoxic than chlorambucil and also a mixture of chlorambucil and the parent organoruthenium RAPTA compound. In a proof-of-principle experiment, the cross-linking of DNA and protein fragments by a chlorambucil-RAPTA derivative was observed.
Asunto(s)
Antineoplásicos Alquilantes/química , Antineoplásicos Alquilantes/farmacología , Clorambucilo/química , Complejos de Coordinación/química , ADN/química , Proteínas/química , Alquilación , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Clorambucilo/farmacología , Complejos de Coordinación/síntesis química , Complejos de Coordinación/farmacología , Cimenos , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Cinética , Células MCF-7 , Compuestos Organometálicos/química , Proteínas/metabolismo , Ubiquitina/química , Ubiquitina/metabolismoRESUMEN
A water-stable phosphoramidate Ru(arene) metallodrug shows antiproliferative activity comparable to KP1019 in human cancer cell lines. This novel compound can cross-link the peptide backbone of cytochrome c, but features low apoptosis inducing properties.