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In the European Union, the Chemicals Strategy for Sustainability (CSS) highlights the need to enhance the identification and assessment of substances of concern while reducing animal testing, thus fostering the development and use of New Approach Methodologies (NAMs) such as in silico, in vitro and in chemico. In the United States, the Tox21 strategy aims at shifting toxicological assessments away from traditional animal studies towards target-specific, mechanism-based and biological observations mainly obtained by using NAMs. Many other jurisdictions around the world are also increasing the use of NAMs. Hence, the provision of dedicated non-animal toxicological data and reporting formats as a basis for chemical risk assessment is necessary. Harmonising data reporting is crucial when aiming at re-using and sharing data for chemical risk assessment across jurisdictions. The OECD has developed a series of OECD Harmonised Templates (OHT), which are standard data formats designed for reporting information used for the risk assessment of chemicals relevant to their intrinsic properties, including effects on human health (e.g., toxicokinetics, skin sensitisation, repeated dose toxicity) and the environment (e.g., toxicity to test species and wildlife, biodegradation in soil, metabolism of residues in crops). The objective of this paper is to demonstrate the applicability of the OHT standard format for reporting information under various chemical risk assessment regimes, and to provide users with practical guidance on the use of OHT 201, in particular to report test results on intermediate effects and mechanistic information.
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Organización para la Cooperación y el Desarrollo Económico , Piel , Humanos , Medición de Riesgo/métodosRESUMEN
Biomedical research on Alzheimer's disease (AD), breast cancer (BC) and prostate cancer (PC) has globally improved our understanding of the etiopathological mechanisms underlying the onset of these diseases, often with the goal to identify associated genetic and environmental risk factors and develop new medicines. However, the prevalence of these diseases and failure rate in drug development remain high. Being able to retrospectively monitor the major scientific breakthroughs and impact of such investment endeavors is important to re-address funding strategies if and when needed. The EU has supported research into those diseases via its successive framework programmes for research, technological development and innovation. The European Commission (EC) has already undertaken several activities to monitor research impact. As an additional contribution, the EC Joint Research Centre (JRC) launched in 2020 a survey addressed to former and current participants of EU-funded research projects in the fields of AD, BC and PC, with the aim to understand how EU-funded research has contributed to scientific innovation and societal impact, and how the selection of the experimental models may have underpinned the advances made. Further feedback was also gathered through in-depth interviews with some selected survey participants representative of the diverse pre-clinical models used in the EU-funded projects. A comprehensive analysis of survey replies, complemented with the information derived from the interviews, has recently been published in a Synopsis report. Here we discuss the main findings of this analysis and propose a set of priority actions that could be considered to help improving the translation of scientific innovation of biomedical research into societal impact.
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Enfermedad de Alzheimer , Investigación Biomédica , Neoplasias de la Mama , Neoplasias de la Próstata , Masculino , Humanos , Estudios RetrospectivosRESUMEN
Low delivery of many anticancer drugs across the blood-brain barrier (BBB) is a limitation to the success of chemotherapy in glioblastoma. This is because of the high levels of ATP-binding cassette transporters like P-glycoprotein (Pgp/ABCB1), which effluxes drugs back to the bloodstream. Temozolomide is one of the few agents able to cross the BBB; its effects on BBB cells permeability and Pgp activity are not known. We found that temozolomide, at therapeutic concentration, increased the transport of Pgp substrates across human brain microvascular endothelial cells and decreased the expression of Pgp. By methylating the promoter of Wnt3 gene, temozolomide lowers the endogenous synthesis of Wnt3 in BBB cells, disrupts the Wnt3/glycogen synthase kinase 3/ß-catenin signaling, and reduces the binding of ß-catenin on the promoter of mdr1 gene, which encodes for Pgp. In co-culture models of BBB cells and human glioblastoma cells, pre-treatment with temozolomide increases the delivery, cytotoxicity, and antiproliferative effects of doxorubicin, vinblastine, and topotecan, three substrates of Pgp that are usually poorly delivered across BBB. Our work suggests that temozolomide increases the BBB permeability of drugs that are normally effluxed by Pgp back to the bloodstream. These findings may pave the way to new combinatorial chemotherapy schemes in glioblastoma.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antineoplásicos/farmacología , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar/efectos de los fármacos , Dacarbazina/análogos & derivados , Regulación de la Expresión Génica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Proteína Wnt3/metabolismo , Línea Celular Tumoral , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Dacarbazina/farmacología , Regulación de la Expresión Génica/fisiología , Humanos , Regiones Promotoras Genéticas/genética , Transducción de Señal/fisiología , Temozolomida , beta Catenina/metabolismoRESUMEN
In this work we investigated how the surface charge and the presence of polyethylene glycol (PEG) on liposome carriers affect the delivery of the encapsulated doxorubicin in P-glycoprotein (Pgp)-overexpressing cells. We found that neutral net charge was critical to favour the liposome uptake and decrease the Vmax of doxorubicin efflux. PEG-coating was necessary to increase the Km of doxorubicin for Pgp. In particular the PEGylated phospholipid present in neutral liposomes, i.e. PEGylated distearoyl-phosphatidylethanolamine (DSPE-PEG), was a Pgp allosteric inhibitor, increased doxorubicin Km and inhibited Pgp ATPase activity. Site-directed mutagenesis experiments suggested that the domain centred around glycine 185 of Pgp was necessary for these inhibitory properties of DSPE-PEG and PEGylated neutral liposomes. We conclude that both surface charge and PEGylation must be considered to optimize the doxorubicin delivery within chemoresistant cells. DSPE-PEG-enriched particles may represent promising tools for therapeutic and diagnostic applications in tissues with high levels of Pgp. FROM THE CLINICAL EDITOR: These authors investigated how surface charge and PEGylation of liposome carriers affect the delivery of encapsulated doxorubicin to Pgp-overexpressing cells, concluding that both factors need to be considered in order to optimize doxorubicin delivery to chemoresistant cells.
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Doxorrubicina/administración & dosificación , Sistemas de Liberación de Medicamentos , Liposomas/administración & dosificación , Neoplasias/tratamiento farmacológico , Subfamilia B de Transportador de Casetes de Unión a ATP/antagonistas & inhibidores , Adenosina Trifosfatasas/metabolismo , Doxorrubicina/química , Resistencia a Antineoplásicos/efectos de los fármacos , Humanos , Liposomas/química , Polietilenglicoles/administración & dosificación , Polietilenglicoles/química , Propiedades de SuperficieRESUMEN
BACKGROUND: The activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors. METHODS: We studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells. RESULTS: MDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells. CONCLUSIONS: Our work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells.
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Membrana Celular/metabolismo , Colesterol/biosíntesis , Neoplasias del Colon/metabolismo , Ácidos Docosahexaenoicos/farmacología , Ácido Eicosapentaenoico/farmacología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Antibióticos Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Ácidos Docosahexaenoicos/metabolismo , Regulación hacia Abajo , Doxorrubicina/uso terapéutico , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Ácido Eicosapentaenoico/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HT29 , Humanos , Fosforilación , UbiquitinaciónRESUMEN
In previous studies, we showed that nitric oxide (NO) donors and synthetic doxorubicins (DOXs) modified with moieties containing NO-releasing groups--such as nitrooxy-DOX (NitDOX) or 3-phenylsulfonylfuroxan-DOX (FurDOX)--overcome drug resistance by decreasing the activity of ATP-binding cassette (ABC) transporters that can extrude the drug. Here, we have investigated the biochemical mechanisms by which NitDOX and FurDOX exert antitumor effects. Both NitDOX and FurDOX were more cytotoxic than DOX against drug-resistant cells. Interestingly, NitDOX exhibited a faster uptake and an extranuclear distribution. NitDOX was preferentially localized in the mitochondria, where it nitrated and inhibited the mitochondria-associated ABC transporters, decreased the flux through the tricarboxylic acid cycle, slowed down the activity of complex I, lowered the synthesis of ATP, induced oxidative and nitrosative stress, and elicited the release of cytochrome c and the activation of caspase-9 and -3 in DOX-resistant cells. We suggest that NitDOX may represent the prototype of a new class of multifunctional anthracyclines, which have cellular targets different from conventional anthracyclines and greater efficacy against drug-resistant tumors.
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Antineoplásicos/farmacología , Doxorrubicina/análogos & derivados , Doxorrubicina/farmacología , Mitocondrias/efectos de los fármacos , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico/farmacología , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Caspasa 3/metabolismo , Caspasa 9/metabolismo , Ciclo del Ácido Cítrico/efectos de los fármacos , Citocromos c/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Resistencia a Antineoplásicos , Complejo I de Transporte de Electrón/metabolismo , Células HT29 , Humanos , Células K562 , Mitocondrias/metabolismo , Óxido Nítrico/metabolismo , Estrés Oxidativo/efectos de los fármacosRESUMEN
Cardioactive glycosides exert positive inotropic effects on cardiomyocytes through the inhibition of Na(+)/K(+)-ATPase. We showed previously that in human hepatoma cells, digoxin and ouabain increase the rate of the mevalonate cascade and therefore have Na(+)/K(+)-ATPase-independent effects. In the present study we found that they increase the expression and activity of 3-hydroxy-3 methylglutaryl-CoA reductase and the synthesis of cholesterol in cardiomyocytes, their main target cells. Surprisingly this did not promote intracellular cholesterol accumulation. The glycosides activated the liver X receptor transcription factor and increased the expression of ABCA1 (ATP-binding cassette protein A1) transporter, which mediates the efflux of cholesterol and its delivery to apolipoprotein A-I. By increasing the synthesis of ubiquinone, another derivative of the mevalonate cascade, digoxin and ouabain simultaneously enhanced the rate of electron transport in the mitochondrial respiratory chain and the synthesis of ATP. Mice treated with digoxin showed lower cholesterol and higher ubiquinone content in their hearts, and a small increase in their serum HDL (high-density lipoprotein) cholesterol. The results of the present study suggest that cardioactive glycosides may have a role in the reverse transport of cholesterol and in the energy metabolism of cardiomyocytes.
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Adenosina Trifosfato/biosíntesis , Colesterol/biosíntesis , Digoxina/farmacología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Receptores Nucleares Huérfanos/fisiología , Ouabaína/farmacología , Transportador 1 de Casete de Unión a ATP , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Apolipoproteína A-I/metabolismo , Línea Celular , Transporte de Electrón/efectos de los fármacos , Hidroximetilglutaril-CoA Reductasas/metabolismo , Lipoproteínas HDL/metabolismo , Receptores X del Hígado , Masculino , Ácido Mevalónico/metabolismo , Ratones , Ratas , Ubiquinona/biosíntesisRESUMEN
In a century where toxicology and chemical risk assessment are embracing alternative methods to animal testing, there is an opportunity to understand the causal factors of neurodevelopmental disorders such as learning and memory disabilities in children, as a foundation to predict adverse effects. New testing paradigms, along with the advances in probabilistic modelling, can help with the formulation of mechanistically-driven hypotheses on how exposure to environmental chemicals could potentially lead to developmental neurotoxicity (DNT). This investigation aimed to develop a Bayesian hierarchical model of a simplified AOP network for DNT. The model predicted the probability that a compound induces each of three selected common key events (CKEs) of the simplified AOP network and the adverse outcome (AO) of DNT, taking into account correlations and causal relations informed by the key event relationships (KERs). A dataset of 88 compounds representing pharmaceuticals, industrial chemicals and pesticides was compiled including physicochemical properties as well as in silico and in vitro information. The Bayesian model was able to predict DNT potential with an accuracy of 76%, classifying the compounds into low, medium or high probability classes. The modelling workflow achieved three further goals: it dealt with missing values; accommodated unbalanced and correlated data; and followed the structure of a directed acyclic graph (DAG) to simulate the simplified AOP network. Overall, the model demonstrated the utility of Bayesian hierarchical modelling for the development of quantitative AOP (qAOP) models and for informing the use of new approach methodologies (NAMs) in chemical risk assessment.
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The adverse outcome pathway (AOP) is a conceptual construct that facilitates organisation and interpretation of mechanistic data representing multiple biological levels and deriving from a range of methodological approaches including in silico, in vitro and in vivo assays. AOPs are playing an increasingly important role in the chemical safety assessment paradigm and quantification of AOPs is an important step towards a more reliable prediction of chemically induced adverse effects. Modelling methodologies require the identification, extraction and use of reliable data and information to support the inclusion of quantitative considerations in AOP development. An extensive and growing range of digital resources are available to support the modelling of quantitative AOPs, providing a wide range of information, but also requiring guidance for their practical application. A framework for qAOP development is proposed based on feedback from a group of experts and three qAOP case studies. The proposed framework provides a harmonised approach for both regulators and scientists working in this area.
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The anticancer drug doxorubicin induces the synthesis of nitric oxide, a small molecule that enhances the drug cytotoxicity and reduces the drug efflux through the membrane pump P-glycoprotein (Pgp). Doxorubicin also induces the translocation on the plasma membrane of the protein calreticulin (CRT), which allows tumour cells to be phagocytized by dendritic cells. We have shown that doxorubicin elicits nitric oxide synthesis and CRT exposure only in drug-sensitive cells, not in drug-resistant ones, which are indeed chemo-immunoresistant. In this work, we investigate the mechanisms by which nitric oxide induces the translocation of CRT and the molecular basis of this chemo-immunoresistance. In the drug-sensitive colon cancer HT29 cells doxorubicin increased nitric oxide synthesis, CRT exposure and cells phagocytosis. Nitric oxide promoted the translocation of CRT in a guanosine monophosphate (cGMP) and actin cytoskeleton-dependent way. CRT translocation did not occur in drug-resistant HT29-dx cells, where the doxorubicin-induced nitric oxide synthesis was absent. By increasing nitric oxide with stimuli other than doxorubicin, the CRT exposure was obtained also in HT29-dx cells. Although in sensitive cells the CRT translocation was followed by the phagocytosis, in drug-resistant cells the phagocytosis did not occur despite the CRT exposure. In HT29-dx cells CRT was bound to Pgp and only by silencing the latter the CRT-operated phagocytosis was restored, suggesting that Pgp impairs the functional activity of CRT and the tumour cells phagocytosis. Our work suggests that the levels of nitric oxide and Pgp critically modulate the recognition of the tumour cells by dendritic cells, and proposes a new potential therapeutic approach against chemo-immunoresistant tumours.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Neoplasias del Colon/metabolismo , Células HT29/metabolismo , Óxido Nítrico/metabolismo , Fagocitosis/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Animales , Antibióticos Antineoplásicos/farmacología , Calreticulina/metabolismo , GMP Cíclico/metabolismo , Citoesqueleto/metabolismo , Células Dendríticas/metabolismo , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Células HT29/efectos de los fármacos , Humanos , Óxido Nítrico Sintasa/metabolismoRESUMEN
Dementia and cancer are becoming increasingly prevalent in Western countries. In the last two decades, research focused on Alzheimer's disease (AD) and cancer, in particular, breast cancer (BC) and prostate cancer (PC), has been substantially funded both in Europe and worldwide. While scientific research outcomes have contributed to increase our understanding of the disease etiopathology, still the prevalence of these chronic degenerative conditions remains very high across the globe. By definition, no model is perfect. In particular, animal models of AD, BC, and PC have been and still are traditionally used in basic/fundamental, translational, and preclinical research to study human disease mechanisms, identify new therapeutic targets, and develop new drugs. However, animals do not adequately model some essential features of human disease; therefore, they are often unable to pave the way to the development of drugs effective in human patients. The rise of new technological tools and models in life science, and the increasing need for multidisciplinary approaches have encouraged many interdisciplinary research initiatives. With considerable funds being invested in biomedical research, it is becoming pivotal to define and apply indicators to monitor the contribution to innovation and impact of funded research. Here, we discuss some of the issues underlying translational failure in AD, BC, and PC research, and describe how indicators could be applied to retrospectively measure outputs and impact of funded biomedical research.
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Digoxin and ouabain are cardioactive glycosides, which inhibit the Na+/K+-ATPase pump and in this way they increase the intracellular concentration of cytosolic calcium ([Ca2+](i)). They are also strong inducers of the P-glycoprotein (Pgp), a transmembrane transporter which extrudes several drugs, including anticancer agents like doxorubicin. An increased amount of Pgp limits the absorption of drugs through epithelial cells, thus inducing resistance to chemotherapy. The mechanism by which cardioactive glycosides increase Pgp is not known and in this work we investigated whether digoxin and ouabain elicited the expression of Pgp with a calcium-driven mechanism. In human colon cancer HT29 cells both glycosides increased the [Ca2+](i) and this event was dependent on the calcium influx via the Na+/Ca2+ exchanger. The increased [Ca2+](i) enhanced the activity of the calmodulin kinase II enzyme, which in turn activated the transcription factor hypoxia-inducible factor-1alpha. This one was responsible for the increased expression of Pgp, which actively extruded doxorubicin from the cells and significantly reduced the pro-apoptotic effect of the drug. All the effects of glycosides were prevented by inhibiting the Na+/Ca2+ exchanger or the calmodulin kinase II. This work clarified the molecular mechanisms by which digoxin and oubain induce Pgp and pointed out that the administration of cardioactive glycosides may widely affect the absorption of drugs in colon epithelia. Moreover, our results suggest that the efficacy of chemotherapeutic agent substrates of Pgp may be strongly reduced in patients taking digoxin.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/biosíntesis , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Neoplasias del Colon/metabolismo , Digoxina/farmacología , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ouabaína/farmacología , Secuencia de Bases , Western Blotting , Neoplasias del Colon/enzimología , Cartilla de ADN , Doxorrubicina/farmacología , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Células HT29 , Humanos , Fosforilación , Reacción en Cadena de la PolimerasaRESUMEN
NAD(P)H oxidases (NOXs) are a family of enzymes catalyzing the univalent reduction of oxygen to produce the superoxide anion radical, which in turn can be converted in other reactive oxygen species (ROS) and may participate to the formation of reactive nitrogen derivatives, such as peroxynitrite. By virtue of their activity, NOXs may represent a double-edged sword for the organism's homeostasis. On one hand ROS participate in host defence by killing invading microbes and may regulate several important physiological functions, such as cell signalling, regulation of cell growth and differentiation, oxygen sensing, angiogenesis, fertilization and control of vascular tone. On the other hand ROS may play an important role in pathological processes such as hypertension, atherosclerosis, diabetes, cancer, ischemia/reperfusion injury, neurodegenerative diseases. Many roles suggested for NOXs in various tissues and physiopathological situations have been inferred by the in vitro and in vivo effects of several NOX inhibitors. In particular, most studies are based on the use of two compounds, diphenyleneiodonium and apocynin. Aim of this review is to describe the main features of these two compounds, to show that they cannot be used as specific NOX inhibitors and to solicit researchers to find other tools for investigating the role of NOXs.
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Inhibidores Enzimáticos/farmacología , NADPH Oxidasas/antagonistas & inhibidores , Acetofenonas/farmacología , Animales , Flavoproteínas/antagonistas & inhibidores , Humanos , Óxido Nítrico/biosíntesis , Compuestos Onio/farmacología , Estrés Oxidativo , Especies de Nitrógeno Reactivo/metabolismo , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Flame retardants (FRs) are a diverse group of chemicals used as additives in a wide range of products to inhibit, suppress, or delay ignition and to prevent the spread of fire. Halogenated FRs (HFRs) are widely used because of their low impact on other material properties and the low loading levels necessary to meet the required flame retardancy. Health and environmental hazards associated with some halogenated FRs have driven research for identifying safer alternatives. A variety of halogen-free FRs are available on the market, including organic (phosphorus and nitrogen based chemicals) and inorganic (metals) materials. Multi-walled carbon nanotubes (MWCNT) have been demonstrated to act as an effective/synergistic co-additive in some FR applications and could thereby contribute to reducing the loading of FRs in products and improving their performance. As part of the FP7 project DEROCA we carried out a chemical alternatives assessment (CAA). This is a methodology for identifying, comparing and selecting safer alternatives to chemicals of concern based on criteria for categorising human and environmental toxicity as well as environmental fate. In the project we assessed the hazard data of different halogen-free FRs to be applied in 5 industrial and consumer products and here we present the results for MWCNT, aluminium diethylphosphinate, aluminium trihydroxide, N-alkoxy hindered amines and red phosphorus compared to the HFR decabromodiphenylether. We consulted the REACH guidance, the criteria of the U.S.-EPA Design for Environment (DfE) and the GreenScreen® Assessment to assess and compare intrinsic properties affecting the hazard potential. A comparison/ranking of exposure reference values such as Derived No Effect Levels (DNELs) showed that FRs of concern are not identified by a low DNEL. A comparison based on hazard designations according to the U.S.-EPA DfE and GreenScreen® for human health endpoints, aquatic toxicity and environmental fate showed that the major differences between FRs of concern and their proposed alternatives are the potential for bioaccumulation and CMR (carcinogenic, mutagenic or reprotoxic) effects. As most alternatives are inorganic chemicals, persistence (alone) is not a suitable criterion. From our experiences in carrying out a CAA we conclude: i) REACH registration dossiers provide a comprehensive source of hazard information for an alternative assessment. It is important to consider that the presented data is subject to changes and its quality is variable. ii) Correct identification of the chemicals is crucial to retrieve the right data. This can be challenging for mixtures, reaction products or nanomaterials or when only trade names are available. iii) The quality of the data and the practice on how to fill data gaps can have a huge impact on the results and conclusions. iv) Current assessment criteria have mainly been developed for organic chemicals and create challenges when applied to inorganic solids, including nanomaterials. It is therefore crucial to analyse and report uncertainties for each decision making step.
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Contaminantes Ambientales/toxicidad , Retardadores de Llama/toxicidad , Nanotubos de Carbono/química , Seguridad de Productos para el Consumidor , Humanos , Medición de RiesgoRESUMEN
INTRODUCTION: The Adverse Outcome Pathway framework is increasingly used to integrate data generated based on traditional and emerging toxicity testing paradigms. As the number of AOP descriptions has increased, so has the need to define the AOP in computable terms. MATERIALS AND METHODS: Herein, we present a comprehensive annotation of 172 AOPs housed in the AOP-Wiki as of December 4, 2016 using terms from existing biological ontologies. RESULTS: AOP Key Events (KEs) were assigned ontology terms using a concept called the Event Component, which consists of a Process, an Object, and an Action term, with each term originating from ontologies and other controlled vocabularies. Annotation of KEs with ontology classes from fourteen ontologies and controlled vocabularies resulted in a total of 685 KEs being annotated with a total of 809 Event Components. A set of seven conventions resulted, defining the annotation of KEs via Event Components. DISCUSSION: This expanded annotation of AOPs allows computational reasoners to aid in both AOP development and applications. In addition, the incorporation of explicit biological objects will reduce the time required for converting a qualitative AOP description into a conceptual model that can support computational modeling. As high throughput genomics becomes a more important part of the high throughput toxicity testing landscape, the new approaches described here for annotating key events will also promote the visualization and analysis of genomics data in an AOP context.
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BACKGROUND: Multidrug resistant cancer cells are hard to eradicate for the inefficacy of conventional anticancer drugs. Besides escaping the cytotoxic effects of chemotherapy, they also bypass the pro-immunogenic effects induced by anticancer drugs: indeed they are not well recognized by host dendritic cells and do not elicit a durable anti-tumor immunity. It has not yet been investigated whether multidrug resistant cells have a different ability to induce immunosuppression than chemosensitive ones. We addressed this issue in human and murine chemosensitive and multidrug resistant cancer cells. RESULTS: We found that the activity and expression of indoleamine 2,3-dioxygenase 1 (IDO1), which catalyzes the conversion of tryptophan into the immunosuppressive metabolite kynurenine, was higher in all the multidrug resistant cells analyzed and that IDO1 inhibition reduced the growth of drug-resistant tumors in immunocompetent animals. In chemoresistant cells the basal activity of JAK1/STAT1 and JAK1/STAT3 signaling was higher, the STAT3 inhibitor PIAS3 was down-regulated, and the autocrine production of STAT3-target and IDO1-inducers cytokines IL-6, IL-4, IL-1ß, IL-13, TNF-α and CD40L, was increased. The disruption of the JAK/STAT signaling lowered the IDO1 activity and reversed the kynurenine-induced pro-immunosuppressive effects, as revealed by the restored proliferation of T-lymphocytes in STAT-silenced chemoresistant cells. CONCLUSIONS: Our work shows that multidrug resistant cells have a stronger immunosuppressive attitude than chemosensitive cells, due to the constitutive activation of the JAK/STAT/IDO1 axis, thus resulting chemo- and immune-evasive. Disrupting this axis may significantly improve the efficacy of chemo-immunotherapy protocols against resistant tumors.
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Citocinas/metabolismo , Janus Quinasa 1/metabolismo , Quinurenina/metabolismo , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT3/metabolismo , Transducción de Señal , Animales , Comunicación Autocrina , Línea Celular Tumoral , Regulación hacia Abajo , Resistencia a Antineoplásicos , Femenino , Células HT29 , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Células K562 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Chaperonas Moleculares/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Proteínas Inhibidoras de STAT Activados/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Factor de Transcripción STAT1/antagonistas & inhibidores , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT3/antagonistas & inhibidores , Factor de Transcripción STAT3/genética , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
Multidrug resistance (MDR) in cancer cells is a challenging phenomenon often associated with P-glycoprotein (Pgp) surface expression. Finding new ways to bypass Pgp-mediated MDR still remains a daunting challenge towards the successful treatment of malignant neoplasms such as colorectal cancer.We applied the Cell Surface Capture technology to chemosensitive and chemoresistant human colon cancer to explore the cell surface proteome of Pgp-expressing cells in a discovery-driven fashion. Comparative quantitative analysis of identified cell surface glycoproteins revealed carbonic anhydrase type XII (CAXII) to be up-regulated on the surface of chemoresistant cells, similarly to Pgp. In cellular models showing an acquired MDR phenotype due to the selective pressure of chemotherapy, the progressive increase of the transcription factor hypoxia-inducible factor-1 alpha was paralleled by the simultaneous up-regulation of Pgp and CAXII. CAXII and Pgp physically interacted at the cell surface. CAXII silencing or pharmacological inhibition with acetazolamide decreased the ATPase activity of Pgp by altering the optimal pH at which Pgp operated and promoted chemosensitization to Pgp substrates in MDR cells.We propose CAXII as a new secondary marker of the MDR phenotype that influences Pgp activity directly and can be used as a pharmacological target for MDR research and potential treatment.
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Antineoplásicos/farmacología , Inhibidores de Anhidrasa Carbónica/farmacología , Anhidrasas Carbónicas/metabolismo , Neoplasias del Colon/tratamiento farmacológico , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Neoplasias Pulmonares/tratamiento farmacológico , Terapia Molecular Dirigida , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Apoptosis/efectos de los fármacos , Anhidrasas Carbónicas/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Proliferación Celular/efectos de los fármacos , Técnicas de Visualización de Superficie Celular , Senescencia Celular/efectos de los fármacos , Neoplasias del Colon/enzimología , Neoplasias del Colon/genética , Neoplasias del Colon/patología , Doxorrubicina/metabolismo , Doxorrubicina/farmacología , Regulación Neoplásica de la Expresión Génica , Células HT29 , Humanos , Concentración de Iones de Hidrógeno , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias Pulmonares/enzimología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Fenotipo , Proteómica/métodos , Interferencia de ARN , Transducción de Señal/efectos de los fármacos , TransfecciónRESUMEN
The human malignant mesothelioma (HMM) is characterized by a chemoresistant and immunosuppressive phenotype. An effective strategy to restore chemosensitivity and immune reactivity against HMM is lacking. We investigated whether the use of zoledronic acid is an effective chemo-immunosensitizing strategy. We compared primary HMM samples with non-transformed mesothelial cells. HMM cells had higher rate of cholesterol and isoprenoid synthesis, constitutive activation of Ras/extracellular signal-regulated kinase1/2 (ERK1/2)/hypoxia inducible factor-1α (HIF-1α) pathway and up-regulation of the drug efflux transporter P-glycoprotein (Pgp). By decreasing the isoprenoid supply, zoledronic acid down-regulated the Ras/ERK1/2/HIF-1α/Pgp axis and chemosensitized the HMM cells to Pgp substrates. The HMM cells also produced higher amounts of kynurenine, decreased the proliferation of T-lymphocytes and expanded the number of T-regulatory (Treg) cells. Kynurenine synthesis was due to the transcription of the indoleamine 1,2 dioxygenase (IDO) enzyme, consequent to the activation of the signal transducer and activator of transcription-3 (STAT3). By reducing the activity of the Ras/ERK1/2/STAT3/IDO axis, zoledronic acid lowered the kyurenine synthesis and the expansion of Treg cells, and increased the proliferation of T-lymphocytes. Thanks to its ability to decrease Ras/ERK1/2 activity, which is responsible for both Pgp-mediated chemoresistance and IDO-mediated immunosuppression, zoledronic acid is an effective chemo-immunosensitizing agent in HMM cells.
Asunto(s)
Difosfonatos/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Imidazoles/farmacología , Neoplasias Pulmonares/tratamiento farmacológico , Mesotelioma/tratamiento farmacológico , Linfocitos T/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Anciano , Anciano de 80 o más Años , Western Blotting , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colesterol/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Indolamina-Pirrol 2,3,-Dioxigenasa/genética , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Quinurenina/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Mesotelioma/genética , Mesotelioma/metabolismo , Mesotelioma Maligno , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Transducción de Señal/efectos de los fármacos , Linfocitos T/patología , Terpenos/metabolismo , Células Tumorales Cultivadas , Ácido Zoledrónico , Proteínas ras/metabolismoRESUMEN
The immunoglobulin heavy-chain variable region (IGHV) mutational status is a strong determinant of remission duration in chronic lymphocytic leukemia (CLL). The aim of this work was to compare the multidrug resistance (MDR) signature of IGHV mutated and unmutated CLL cells, identifying biochemical and molecular targets potentially amenable to therapeutic intervention.We found that the mevalonate pathway-dependent Ras/ERK1-2 and RhoA/RhoA kinase signaling cascades, and the downstream HIF-1α/P-glycoprotein axis were more active in IGHV unmutated than in mutated cells, leading to a constitutive protection from doxorubicin-induced cytotoxicity. The constitutive MDR phenotype of IGHV unmutated cells was partially dependent on B cell receptor signaling, as shown by the inhibitory effect exerted by ibrutinib. Stromal cells further protected IGHV unmutated cells from doxorubicin by upregulating Ras/ERK1-2, RhoA/RhoA kinase, Akt, HIF-1α and P-glycoprotein activities. Mevalonate pathway inhibition with simvastatin abrogated these signaling pathways and reversed the resistance of IGHV unmutated cells to doxorubicin, also counteracting the protective effect exerted by stromal cells. Similar results were obtained via the targeted inhibition of the downstream molecules ERK1-2, RhoA kinase and HIF-1α.Therefore, targeting the mevalonate pathway and its downstream signaling cascades is a promising strategy to circumvent the MDR signature of IGHV unmutated CLL cells.
Asunto(s)
Doxorrubicina/farmacología , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Simvastatina/farmacología , Células del Estroma/efectos de los fármacos , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Animales , Antibióticos Antineoplásicos/farmacología , Western Blotting , Línea Celular , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Resistencia a Múltiples Medicamentos/genética , Femenino , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/sangre , Leucemia Linfocítica Crónica de Células B/genética , Leucemia Linfocítica Crónica de Células B/patología , Masculino , Persona de Mediana Edad , Mutación , Transducción de Señal/efectos de los fármacos , Células del Estroma/citología , Células del Estroma/metabolismo , Células Tumorales CultivadasRESUMEN
The blood-brain barrier (BBB), the unusual microvascular endothelial interface between the central nervous system (CNS) and the circulatory system, is a major hindrance to drug delivery in the brain parenchyma. Besides the absence of fenestrations and the abundance of tight junctions, ATP-binding cassette (ABC) transporters critically reduce drug entry within the CNS, as they carry many drugs back into the bloodstream. Nanoparticle- and liposome-carried drugs, because of their increased cellular uptake and reduced efflux through ABC transporters, have been developed in recent times to circumvent the low drug permeability of the BBB. This review discusses the role of ABC transporters in controlling drug penetration into the brain parenchyma, the rationale for using nanoparticle- and liposome-based strategies to increase drug delivery across the BBB and new therapeutic strategies for using nanoparticle- and liposome-carried drugs in different conditions, ranging from CNS tumors and neurodegenerative diseases to viral infections and epilepsy.