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N-Nitrosamine impurities, including nitrosamine drug substance-related impurities (NDSRIs), have challenged pharmaceutical industry and regulators alike and affected the global drug supply over the past 5 years. Nitrosamines are a class of known carcinogens, but NDSRIs have posed additional challenges as many lack empirical data to establish acceptable intake (AI) limits. Read-across analysis from surrogates has been used to identify AI limits in some cases; however, this approach is limited by the availability of robustly-tested surrogates matching the structural features of NDSRIs, which usually contain a diverse array of functional groups. Furthermore, the absence of a surrogate has resulted in conservative AI limits in some cases, posing practical challenges for impurity control. Therefore, a new framework for determining recommended AI limits was urgently needed. Here, the Carcinogenic Potency Categorization Approach (CPCA) and its supporting scientific rationale are presented. The CPCA is a rapidly-applied structure-activity relationship-based method that assigns a nitrosamine to 1 of 5 categories, each with a corresponding AI limit, reflecting predicted carcinogenic potency. The CPCA considers the number and distribution of α-hydrogens at the N-nitroso center and other activating and deactivating structural features of a nitrosamine that affect the α-hydroxylation metabolic activation pathway of carcinogenesis. The CPCA has been adopted internationally by several drug regulatory authorities as a simplified approach and a starting point to determine recommended AI limits for nitrosamines without the need for compound-specific empirical data.
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Carcinógenos , Contaminação de Medicamentos , Nitrosaminas , Nitrosaminas/análise , Nitrosaminas/toxicidade , Carcinógenos/análise , Carcinógenos/toxicidade , Contaminação de Medicamentos/prevenção & controle , Humanos , Animais , Relação Estrutura-Atividade , Medição de Risco , Testes de CarcinogenicidadeRESUMO
Currently the only methods for non-genotoxic carcinogenic hazard assessment accepted by most regulatory authorities are lifetime carcinogenicity studies. However, these involve the use of large numbers of animals and the relevance of their predictive power and results has been scientifically challenged. With increased availability of innovative test methods and enhanced understanding of carcinogenic processes, it is believed that tumour formation can now be better predicted using mechanistic information. A workshop organised by the European Partnership on Alternative Approaches to Animal Testing brought together experts to discuss an alternative, mechanism-based approach for cancer risk assessment of agrochemicals. Data from a toolbox of test methods for detecting modes of action (MOAs) underlying non-genotoxic carcinogenicity are combined with information from subchronic toxicity studies in a weight-of-evidence approach to identify carcinogenic potential of a test substance. The workshop included interactive sessions to discuss the approach using case studies. These showed that fine-tuning is needed, to build confidence in the proposed approach, to ensure scientific correctness, and to address different regulatory needs. This novel approach was considered realistic, and its regulatory acceptance and implementation can be facilitated in the coming years through continued dialogue between all stakeholders and building confidence in alternative approaches.
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Agroquímicos/efeitos adversos , Alternativas aos Testes com Animais , Testes de Carcinogenicidade , Transformação Celular Neoplásica/induzido quimicamente , Neoplasias/induzido quimicamente , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Congressos como Assunto , Humanos , Testes de Mutagenicidade , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Medição de Risco , Testes de Toxicidade Subcrônica , ToxicocinéticaRESUMO
Methadone is an analgesic drug used for pain treatment and heroin substitution. Recently, methadone has been proposed to be useful also for cancer therapy, including glioblastoma multiforme (GBM), the most severe form of brain cancer, because experiments on cultured glioma cells treated with doxorubicin showed promising results. Doxorubicin, however, is not used first-line in GBM therapy. Therefore, we analyzed the cytotoxic effect of methadone alone and in combination with temozolomide, a DNA-alkylating drug that is first-line used in GBM treatment, utilizing GBM-derived cell lines and a human fibroblast cell line. We show that methadone is cytotoxic on its own, inducing apoptosis and necrosis, which was observed at a concentration above 20 µg/mL. Methadone was similar toxic in isogenic MGMT expressing and non-expressing cells, and in LN229 glioblastoma and VH10T human fibroblasts. The apoptosis-inducing activity of methadone is not bound on the opioid receptor (OR), since naloxone, a competitive inhibitor of OR, did not attenuate methadone-induced apoptosis/necrosis. Administrating methadone and temozolomide together, temozolomide had no impact on methadone-induced apoptosis (which occurred 3 days after treatment), while temozolomide-induced apoptosis (which occurred 5 days after treatment) was unaffected at low (non-toxic) methadone concentration (5 µg/mL), and at high (toxic) methadone concentration (20 µg/mL) the cytotoxic effects of methadone and temozolomide were additive. Methadone is not genotoxic, as revealed by comet and γH2AX assay, and did not ameliorate the genotoxic effect of temozolomide. Further, methadone did not induce cellular senescence and had no effect on temozolomide-induced senescence. Although methadone was toxic on senescent cells, it cannot be considered a senolytic drug since cytotoxicity was not specific for senescent cells. Finally, we show that methadone had no impact on the MGMT promoter methylation. Overall, the data show that methadone on glioblastoma cells in vitro is cytotoxic and induces apoptosis/necrosis at doses that are above the level that can be achieved in vivo. It is not genotoxic, and does not ameliorate the cell killing or the senescence-inducing effect of temozolomide (no synergistic effect), indicating it has no impact on temozolomide-induced signaling pathways. The data do not support the notion that concomitant methadone treatment supports temozolomide-based chemotherapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Senescência Celular/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Linhagem Celular Tumoral , Citotoxinas/farmacologia , Metilação de DNA/efeitos dos fármacos , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA de Neoplasias/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Metadona/farmacologia , Regiões Promotoras Genéticas , Temozolomida/farmacologia , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND: Intrinsic chemoresistance of glioblastoma (GBM) is frequently owed to activation of the PI3K and MEK/ERK pathways. These signaling cascades are tightly interconnected however the quantitative contribution of both to intrinsic resistance is still not clear. Here, we aimed at determining the activation status of these pathways in human GBM biopsies and cells and investigating the quantitative impact of both pathways to chemoresistance. METHODS: Receptor tyrosine kinase (RTK) pathways in temozolomide (TMZ) treatment naive or TMZ resistant human GBM biopsies and GBM cells were investigated by proteome profiling and immunoblotting of a subset of proteins. Resistance to drugs and RTK pathway inhibitors was assessed by MTT assays. Apoptotic rates were determined by Annexin V staining and DNA damage with comet assays and immunoblotting. RESULTS: We analyzed activation of RTK pathways by proteome profiling of tumor samples of patients which were diagnosed a secondary GBM and underwent surgery and patients which underwent a second surgery after TMZ treatment due to recurrence of the tumor. We observed substantial activation of the PI3K and MEK/ERK pathways in both groups. However, AKT and CREB phosphorylation was reduced in biopsies of resistant tumors while ERK phosphorylation remained unchanged. Subsequent proteome profiling revealed that multiple RTKs and downstream targets are also activated in three GBM cell lines. We then systematically describe a mechanism of resistance of GBM cell lines and human primary GBM cells to the alkylating drugs TMZ and cisplatin. No specific inhibitor of the upstream RTKs sensitized cells to drug treatment. In contrast, we were able to restore sensitivity to TMZ and cisplatin by inhibiting PI3K in all cell lines and in human primary GBM cells. Interestingly, an opposite effect was observed when we inhibited the MEK/ERK signaling cascade with two different inhibitors. CONCLUSIONS: Temozolomide treatment naive and TMZ resistant GBM biopsies show a distinct activation pattern of the MEK/ERK and PI3K signaling cascades indicating a role of these pathways in resistance development. Both pathways are also activated in GBM cell lines, however, only the PI3K pathway seems to play a crucial role in resistance to alkylating agents and might serve as drug target for chemosensitization.
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Thioredoxin (Trx) overexpression is known to be a cause of chemotherapy resistance in various tumor entities. However, Trx effects on resistance are complex and depend strictly on tissue type. In the present study, we analyzed the impact of the Trx system on intrinsic chemoresistance of human glioblastoma multiforme (GBM) cells to cytostatic drugs. Resistance of GBM cell lines and primary cells to drugs and signaling inhibitors was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. Impact of Trx inhibition on apoptosis was investigated by proteome profiling of a subset of proteins and annexin V apoptosis assays. Trx-interacting protein (TXNIP) was overexpressed by transfection and protein expression was determined by immunoblotting. Pharmacological inhibition of Trx by 1-methyl-2-imidazolyl-disulfide (PX-12) reduced viability of three GBM cell lines, induced expression of active caspase-3, and reduced phosphorylation of AKT-kinase and expression of ß-catenin. Sensitivity to cisplatin could be restored by both PX-12 and recombinant expression of the upstream Trx inhibitor TXNIP, respectively. In addition, PX-12 also sensitized primary human GBM cells to temozolomide. Combined inhibition of Trx and the phosphatidylinositide 3-kinase (PI3K) pathway resulted in massive cell death. We conclude that the Trx system and the PI3K pathway act as a sequential cascade and could potentially present a new drug target.
Assuntos
Apoptose/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Citostáticos/farmacologia , Tiorredoxinas/metabolismo , Western Blotting , Proteínas de Transporte/antagonistas & inibidores , Linhagem Celular Tumoral , Dissulfetos/farmacologia , Glioblastoma/metabolismo , Glioma/metabolismo , Humanos , Imidazóis/farmacologia , Modelos Biológicos , Temozolomida/farmacologia , Tiorredoxinas/antagonistas & inibidoresRESUMO
Narrow Therapeutic Index Drugs (NTIDs) are characterized by a small range between therapeutic and toxicological effect. Missing international harmonized definition for NTIDs the EMA does not even have a definition of NTIDs in contrast to the U.S. FDA, Health Canada, and the Japanese NIHS. Sunitinib, a tyrosine kinase inhibitor (TKI), indicated for the treatment of certain cancer types, will be running off-patent soon. Falling into the category of NTID would have a major impact on regulatory requirements for generic applications. Our analyses of metadata revealed numerous arguments in favor of a NTID designation. We used in vitro experiments to also give initial experimental answers. Five cell types of different tissue origin were examined for determination of IC50-values in cell viability assays. For comparison, the first-in-class TKI Imatinib was used as reference non-NTID drug. In addition, apoptotic proteins were investigated with respect to their expression and phosphorylation status. These in vitro experiments showed systematically higher toxicity of Sunitinib compared to Imatinib and a different expression and phosphorylation pattern of apoptotic proteins. In vitro data can only give preliminary results and further experiments with clinical blood samples and tumor biopsies are needed to finally clarify NTID status of Sunitinib.
Assuntos
Antineoplásicos/toxicidade , Medicamentos Genéricos/toxicidade , Mesilato de Imatinib/toxicidade , Indóis/toxicidade , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/toxicidade , Pirróis/toxicidade , Testes de Toxicidade/métodos , Animais , Antineoplásicos/administração & dosagem , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Controle de Medicamentos e Entorpecentes , Medicamentos Genéricos/administração & dosagem , Humanos , Mesilato de Imatinib/administração & dosagem , Indóis/administração & dosagem , Concentração Inibidora 50 , Neoplasias/enzimologia , Neoplasias/patologia , Especificidade de Órgãos , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Pirróis/administração & dosagem , Medição de Risco , Transdução de Sinais/efeitos dos fármacos , SunitinibeRESUMO
With more than half a billion individuals affected worldwide, obesity has reached pandemic proportions. Development of "brown-like" or "brite" adipocytes within white adipose tissue (WAT) has potential antiobesity and insulin-sensitizing effects. We investigated the role of cyclic GMP (cGMP) signaling, focusing on cGMP-dependent protein kinase I (PKGI) in WAT. PKGI is expressed in murine WAT, primary adipocytes, and 3T3-L1. Treatment of adipocytes with cGMP resulted in increased adipogenesis, with a 54% increase in expression of peroxisome proliferator-activated receptor-γ. Lentiviral overexpression of PKGI further increased adipogenesis, whereas loss of PKGI significantly reduced adipogenic differentiation. In addition to adipogenic effects, PKGI had an antihypertrophic and anti-inflammatory effect via RhoA phosphorylation and reduction of proinflammatory adipokine expression. Moreover, PKGI induced a 4.3-fold increase in abundance of UCP-1 and the development of a brown-like thermogenic program in primary adipocytes. Notably, treatment of C57BL/6 mice with phosphodiesterase inhibitor sildenafil (12 mg/kg/d) for 7 d caused 4.6-fold increase in uncoupling protein-1 expression and promoted establishment of a brown fat cell-like phenotype ("browning") of WAT in vivo. Taken together, PKGI is a key regulator of cell size, adipokine secretion and browning of white fat depots and thus could be a valuable target in developing novel treatments for obesity.
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Adipogenia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , GMP Cíclico/metabolismo , Células 3T3-L1/citologia , Adipócitos/metabolismo , Tecido Adiposo Marrom/citologia , Tecido Adiposo Branco/citologia , Animais , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Canais Iônicos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Obesidade/metabolismo , Transdução de Sinais , Proteína Desacopladora 1RESUMO
INTRODUCTION: GnRH (gonadotropin-releasing hormone) analogues are long-term known to be safe and effective in the clinical management of hormone-dependent advanced prostate cancer. However, their unusual mechanism of action of de-sensitizing pituitary receptors makes generic market entry challenging. In addition, safety aspects like initial flare-up, breakthrough escape, and miniflares render planning and organization of clinical registration trials a complex project. REGULATORY REQUIREMENTS THERAPEUTIC EQUIVALENCE: Regulatory requirements are high as these medicines are compared to bilateral surgical castration with a 100% success rate. GnRH analogues will be used probably even wider in the near future due to demographic development and extension of indications. However, they are challenged by their antagonistic counterparts, which are avoiding flare-up phenomena. The following article deals with regulatory requirements of GnRH analogues in regard to their clinical characteristics.
Assuntos
Antineoplásicos Hormonais/uso terapêutico , Aprovação de Drogas/legislação & jurisprudência , Medicamentos Genéricos/uso terapêutico , Hormônio Liberador de Gonadotropina/uso terapêutico , Neoplasias Hormônio-Dependentes/tratamento farmacológico , Neoplasias da Próstata/tratamento farmacológico , Animais , Antineoplásicos Hormonais/efeitos adversos , Antineoplásicos Hormonais/farmacocinética , Medicamentos Genéricos/efeitos adversos , Medicamentos Genéricos/farmacocinética , Hormônio Liberador de Gonadotropina/efeitos adversos , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/farmacocinética , Humanos , Masculino , Neoplasias Hormônio-Dependentes/metabolismo , Neoplasias Hormônio-Dependentes/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Equivalência Terapêutica , Resultado do TratamentoRESUMO
OBJECTIVE: Sulfonylureas (SUs) are still among the mostly prescribed antidiabetic drugs with an established mode of action: release of insulin from pancreatic ß-cells. In addition, effects of SUs on adipocytes by activation of the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) have been described, which might explain their insulin-sensitizing potential observed in patients. However, there is a discrepancy between the impact of SUs on antidiabetic action and their rather moderate in vitro effect on PPARγ transcriptional activity. Recent studies have shown that some PPARγ ligands can improve insulin sensitivity by blocking PPARγ Ser-273 phosphorylation without having full agonist activity. It is unknown if SUs elicit their antidiabetic effects on adipocytes by inhibition of PPARγ phosphorylation. Here, we investigated if binding of SUs to PPARγ can interfere with PPARγ Ser-273 phosphorylation and determined their antidiabetic actions in vitro in primary human white adipocytes and in vivo in high-fat diet (HFD) obese mice. METHODS: Primary human white preadipocytes were differentiated in the presence of glibenclamide, glimepiride and PPARγ ligands rosiglitazone and SR1664 to compare PPARγ Ser-273 phosphorylation, glucose uptake and adipokine expression. Transcriptional activity at PPARγ was determined by luciferase assays, quantification of PPARγ Ser-273 phosphorylation was determined by Western blotting and CDK5 kinase assays. In silico modelling was performed to gain insight into the binding characteristics of SUs to PPARγ. HFD mice were administered SUs and rosiglitazone for 6 days. PPARγ Ser-273 phosphorylation in white adipose tissue (WAT), body composition, glucose tolerance, adipocyte morphology and expression levels of genes involved in PPARγ activity in WAT and brown adipose tissue (BAT) were evaluated. RESULTS: SUs inhibit phosphorylation of PPARγ at Ser-273 in primary human white adipocytes and exhibit a positive antidiabetic expression profile, which is characterized by up regulation of insulin-sensitizing and down regulation of insulin resistance-inducing adipokines. We demonstrate that SUs directly bind to PPARγ by in silico modelling and inhibit phosphorylation in kinase assays to a similar extend as rosiglitazone and SR1664. In HFD mice SUs reduce PPARγ phosphorylation in WAT and have comparable effects on gene expression to rosiglitazone. In BAT SUs increase UCP1 expression and reduce lipid droplets sizes. CONCLUSIONS: Our findings indicate that a part of SUs extra-pancreatic effects on adipocytes in vitro and in vivo is probably mediated via their interference with PPARγ phosphorylation rather than via classical agonistic activity at clinical concentrations.
Assuntos
Adipócitos , Hipoglicemiantes , PPAR gama , Compostos de Sulfonilureia , PPAR gama/metabolismo , Animais , Fosforilação , Hipoglicemiantes/farmacologia , Camundongos , Compostos de Sulfonilureia/farmacologia , Humanos , Adipócitos/metabolismo , Adipócitos/efeitos dos fármacos , Masculino , Serina/metabolismo , Camundongos Endogâmicos C57BL , Dieta Hiperlipídica/efeitos adversos , Células Cultivadas , Resistência à InsulinaRESUMO
The modulatory interactions between neurotensin (NT) and the dopaminergic neurotransmitter system in the brain suggest that NT may be associated with the progression of Parkinson's disease (PD). NT exerts its neurophysiological effects by interactions with the human NT receptors type 1 (hNTS1) and 2 (hNTS2). Therefore, both receptor subtypes are promising targets for the development of novel NT-based analogs for the treatment of PD. In this study, we used a virtually guided molecular modeling approach to predict the activity of NT(8-13) analogs by investigating the docking models of ligands designed for binding to the human NTS1 and NTS2 receptors. The importance of the residues at positions 8 and/or 9 for hNTS1 and hNTS2 receptor binding affinity was experimentally confirmed by radioligand binding assays. Further in vitro ADME profiling and in vivo studies revealed that, compared to the parent peptide NT(8-13), compound 10 exhibited improved stability and BBB permeability combined with a significant enhancement of the motor function and memory in a mouse model of PD. The herein reported NTS1/NTS2 dual-specific NT(8-13) analogs represent an attractive tool for the development of therapeutic strategies against PD and potentially other CNS disorders.
Assuntos
Neurotensina , Doença de Parkinson , Animais , Humanos , Camundongos , Dopamina , Ligantes , Neurotensina/farmacologia , Neurotensina/metabolismo , Doença de Parkinson/tratamento farmacológico , Ligação Proteica , Receptores de Neurotensina/metabolismoRESUMO
PURPOSE: D,L-methadone (MET), an analgesic drug used for pain treatment and opiate addiction, has achieved attention from oncologists and social media as possible chemoensitizing agent in cancer therapy, notably brain cancer (glioblastoma multiforme, GBM). MET has been reported to enhance doxorubicin-induced cytotoxicity in GBM cells via activation of the µ-opioid receptor (MOR). Here, we extended this work and quantified the toxic effect of MET in comparison to other opioids alone and in combination with doxorubicin and the clinically more relevant alkylating drug temozolomide (TMZ), using a set of GBM cell lines and primary GBM cells. METHODS: MOR expression in GBM cells was investigated by immunofluorescence and immunoblotting. Resistance to drugs alone and in combination with anticancer drugs was assessed by MTT assays. Concentration effect curves were fitted by nonlinear regression analysis and IC50 values were calculated. Apoptosis and necrosis rates were determined by annexin V/propidium iodide (PI)-flow cytometry. RESULTS: MET alone was cytotoxic in all GBM cell lines and primary GBM cells at high micromolar concentrations (IC50 ~ 60-130 µM), observed both in the metabolic MTT assay and by quantifying apoptosis and necrosis, while morphine and oxycodone were not cytotoxic in this concentration range. Naloxone was not able to block MET-induced cytotoxicity, indicating that cell death-inducing effects of MET are not MOR-dependent. We recorded doxorubicin and TMZ concentration- response curves in combination with fixed MET concentrations. MET enhanced doxorubicin-induced cytotoxicity in only one cell line, and in primary cells it was observed only in a particular MET concentration range. In all assays, MET was not effective in sensitizing cells to TMZ. In two cell lines, MET even decreased the cell's sensitivity to TMZ. CONCLUSION: MET was found to be cytotoxic in GBM cells in vitro only at high, clinically not relevant concentrations, where it was effective in inducing apoptosis and necrosis. Sensitizing effects were only observed in combination with doxorubicin, but not with TMZ, and are dependent on cell line and the applied drug concentration. Therefore, our findings do not support the use of MET in the treatment of GBM in combination with TMZ, as no sensitizing effect of MET was observed.
Assuntos
Analgésicos Opioides/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Doxorrubicina/farmacologia , Glioblastoma/tratamento farmacológico , Metadona/farmacologia , Analgésicos Opioides/administração & dosagem , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Doxorrubicina/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Metadona/administração & dosagem , Morfina/farmacologia , Naloxona/farmacologia , Oxicodona/farmacologia , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inibidores , Receptores Opioides mu/biossíntese , Células Tumorais CultivadasRESUMO
OBJECTIVE: Obesity is an enormous burden for patients and health systems world-wide. Brown adipose tissue dissipates energy in response to cold and has been shown to be metabolically active in human adults. The type I transforming growth factor ß (TGFß) receptor Activin receptor-like kinase 7 (Alk7) is highly expressed in adipose tissues and is down-regulated in obese patients. Here, we studied the function of Alk7 in brown adipocytes. METHODS: Using pharmacological and genetic tools, Alk7 signaling pathway and its effects were studied in murine brown adipocytes. Brown adipocyte differentiation and activation was analyzed. RESULTS: Alk7 is highly upregulated during differentiation of brown adipocytes. Interestingly, Alk7 expression is increased by cGMP/protein kinase G (PKG) signaling, which enhances brown adipocyte differentiation. Activin AB effectively activates Alk7 and SMAD3 signaling. Activation of Alk7 in brown preadipocytes suppresses the master adipogenic transcription factor PPARγ and differentiation. Stimulation of Alk7 during late differentiation of brown adipocytes reduces lipid content and adipogenic marker expression but enhances UCP1 expression. CONCLUSIONS: We found a so far unknown crosstalk between cGMP and Alk7 signaling pathways. Tight regulation of Alk7 is required for efficient differentiation of brown adipocytes. Alk7 has differential effects on adipogenic differentiation and the development of the thermogenic program in brown adipocytes.
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Cushing's disease (CD) in a stricter sense derives from pathologic adrenocorticotropic hormone (ACTH) secretion usually triggered by micro- or macroadenoma of the pituitary gland. It is, thus, a form of secondary hypercortisolism. In contrast, Cushing's syndrome (CS) describes the complexity of clinical consequences triggered by excessive cortisol blood levels over extended periods of time irrespective of their origin. CS is a rare disease according to the European orphan regulation affecting not more than 5/10,000 persons in Europe. CD most commonly affects adults aged 20-50 years with a marked female preponderance (1:5 ratio of male vs. female). Patient presentation and clinical symptoms substantially vary depending on duration and plasma levels of cortisol. In 80% of cases CS is ACTH-dependent and in 20% of cases it is ACTH-independent, respectively. Endogenous CS usually is a result of a pituitary tumor. Clinical manifestation of CS, apart from corticotropin-releasing hormone (CRH-), ACTH-, and cortisol-producing (malign and benign) tumors may also be by exogenous glucocorticoid intake. Diagnosis of hypercortisolism (irrespective of its origin) comprises the following: Complete blood count including serum electrolytes, blood sugar etc., urinary free cortisol (UFC) from 24 h-urine sampling and circadian profile of plasma cortisol, plasma ACTH, dehydroepiandrosterone, testosterone itself, and urine steroid profile, Low-Dose-Dexamethasone-Test, High-Dose-Dexamethasone-Test, after endocrine diagnostic tests: magnetic resonance imaging (MRI), ultra-sound, computer tomography (CT) and other localization diagnostics. First-line therapy is trans-sphenoidal surgery (TSS) of the pituitary adenoma (in case of ACTH-producing tumors). In patients not amenable for surgery radiotherapy remains an option. Pharmacological therapy applies when these two options are not amenable or refused. In cases when pharmacological therapy becomes necessary, Pasireotide should be used in first-line in CD. CS patients are at an overall 4-fold higher mortality rate than age- and gender-matched subjects in the general population. The following article describes the most prominent substances used for clinical management of CS and gives a systematic overview of safety profiles, pharmacokinetic (PK)-parameters, and regulatory framework.
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Síndrome de Cushing/tratamento farmacológico , Corticosteroides/uso terapêutico , Síndrome de Cushing/epidemiologia , HumanosRESUMO
Over the last decades, billions have been spent and huge efforts have been taken in basic and clinical cancer research [CA Cancer J Clin63:11-30]. About a decade ago, the arms race between drugs and cancer cells reached a new level by introduction of tyrosine kinase inhibitors (TKI) into pharmacological anti-cancer therapy. According to their molecular mechanism of action, TKI in contrast to so-called "classic" or "conventional" cytostatics belong to the group of targeted cancer medicines, characterized by accurately fitting with biological structures (i.e. active centers of kinases). Numerous (partly orphan) indications are covered by this new class of substances. Approximately ten years after the first substances of this class of medicines were authorized, patent protection will end within the next years. The following article covers clinical meaning and regulatory status of anti-cancer TKI and gives an outlook to what is expected from the introduction of generic anti-cancer TKI.
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Antineoplásicos/farmacologia , Medicamentos Genéricos/farmacologia , Neoplasias/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Antineoplásicos/efeitos adversos , Antineoplásicos/uso terapêutico , Área Sob a Curva , Controle de Medicamentos e Entorpecentes , Medicamentos Genéricos/efeitos adversos , Medicamentos Genéricos/uso terapêutico , Humanos , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/efeitos adversos , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
Obesity has reached pandemic dimensions with more than half a billion adults affected worldwide. Detailed knowledge of adipose biology is required for the development of urgently needed novel therapies directed against obesity. Two types of adipose tissue can be distinguished in humans and mice: white adipose tissue (WAT), which primarily stores energy in the form of lipids and has endocrine functions. In contrast, brown adipose tissue (BAT) dissipates energy in the form of heat (thermogenesis). Recent studies in humans demonstrated that BAT not only plays a role for non-shivering thermogenesis in newborns but is also metabolically active in adults. Here, we describe protocols for the generation of cellular models for the analysis of adipogenesis as well as function of brown and white fat. These models are based on the in vitro differentiation of mesenchymal stem cells (MSCs) isolated from adipose tissues. Using specific differentiation protocols, the role of cGMP signaling in both brown as well as white adipocytes can be studied.
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Adipócitos/metabolismo , GMP Cíclico/metabolismo , Transdução de Sinais , Adipócitos/citologia , Adipócitos Marrons/citologia , Adipócitos Marrons/metabolismo , Adipócitos Brancos/citologia , Adipócitos Brancos/metabolismo , Adipogenia/fisiologia , Animais , Diferenciação Celular , Separação Celular/métodos , GMP Cíclico/química , Ativação Enzimática , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismoRESUMO
Brown adipocytes are a primary site of energy expenditure and reside not only in classical brown adipose tissue but can also be found in white adipose tissue. Here we show that microRNA 155 is enriched in brown adipose tissue and is highly expressed in proliferating brown preadipocytes but declines after induction of differentiation. Interestingly, microRNA 155 and its target, the adipogenic transcription factor CCAAT/enhancer-binding protein ß, form a bistable feedback loop integrating hormonal signals that regulate proliferation or differentiation. Inhibition of microRNA 155 enhances brown adipocyte differentiation and induces a brown adipocyte-like phenotype ('browning') in white adipocytes. Consequently, microRNA 155-deficient mice exhibit increased brown adipose tissue function and 'browning' of white fat tissue. In contrast, transgenic overexpression of microRNA 155 in mice causes a reduction of brown adipose tissue mass and impairment of brown adipose tissue function. These data demonstrate that the bistable loop involving microRNA 155 and CCAAT/enhancer-binding protein ß regulates brown lineage commitment, thereby, controlling the development of brown and beige fat cells.
Assuntos
Adipócitos Marrons/citologia , Adipócitos Brancos/citologia , Diferenciação Celular/genética , Retroalimentação Fisiológica , MicroRNAs/metabolismo , Adipócitos Marrons/metabolismo , Adipócitos Brancos/metabolismo , Adipogenia/genética , Tecido Adiposo Marrom/citologia , Tecido Adiposo Marrom/metabolismo , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Modelos Biológicos , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/metabolismoRESUMO
Two different types of adipose tissues can be found in humans enabling them to respond to starvation and cold: white adipose tissue (WAT) is generally known and stores excess energy in the form of triacylglycerol (TG), insulates against cold, and serves as a mechanical cushion. Brown adipose tissue (BAT) helps newborns to cope with cold. BAT has the capacity to uncouple the mitochondrial respiratory chain, thereby generating heat rather than adenosine triphosphate (ATP). The previously widely held view was that BAT disappears rapidly after birth and is no longer present in adult humans. Using positron emission tomography (PET), however, it was recently shown that metabolically active BAT occurs in defined regions and scattered in WAT of the adult and possibly has an influence on whole-body energy homeostasis. In obese individuals adipose tissue is at the center of metabolic syndrome. Targeting of WAT by thiazolidinediones (TZDs), activators of peroxisome proliferator-activated receptor γ (PPARγ) a 'master' regulator of fat cell biology, is a current therapy for the treatment of type 2 diabetes. Since its unique capacity to increase energy consumption of the body and to dissipate surplus energy as heat, BAT offers new perspectives as a therapeutic target for the treatment of obesity and associated diseases such as type 2 diabetes and metabolic syndrome. Recent discoveries of new signaling pathways of BAT development give rise to new therapeutic possibilities in order to influence BAT content and activity.
RESUMO
Mechanical stimulation is crucial for bone growth and remodeling, and fluid shear stress promotes anabolic responses in osteoblasts through multiple second messengers, including nitric oxide (NO). NO triggers production of cyclic guanosine 3',5'-monophosphate (cGMP), which in turn activates protein kinase G (PKG). We found that the NO-cGMP-PKG signaling pathway activates Src in mechanically stimulated osteoblasts to initiate a proliferative response. PKGII was necessary for Src activation, a process that also required the interaction of Src with ß3 integrins and dephosphorylation of Src by a complex containing the phosphatases SHP-1 (Src homology 2 domain-containing tyrosine phosphatase 1) and SHP-2. PKGII directly phosphorylated and stimulated SHP-1 activity, and fluid shear stress triggered the recruitment of PKGII, Src, SHP-1, and SHP-2 to a mechanosome containing ß3 integrins. PKGII-null mice showed defective Src and ERK (extracellular signal-regulated kinase) signaling in osteoblasts and decreased ERK-dependent gene expression in bone. Our findings reveal a convergence of NO-cGMP-PKG and integrin signaling and establish a previously unknown mechanism of Src activation. These results support the use of PKG-activating drugs to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Mecanotransdução Celular/fisiologia , Osteoblastos/fisiologia , Sistemas do Segundo Mensageiro/fisiologia , Quinases da Família src/metabolismo , Análise de Variância , Animais , Fenômenos Biomecânicos , Bromodesoxiuridina , Fracionamento Celular , Linhagem Celular , Proliferação de Células , Proteínas Quinases Dependentes de GMP Cíclico/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Imunofluorescência , Immunoblotting , Imunoprecipitação , Cadeias beta de Integrinas/metabolismo , Camundongos , Camundongos Knockout , Modelos Biológicos , Óxido Nítrico/metabolismo , Osteoblastos/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estresse MecânicoRESUMO
High malignancy and early metastasis are hallmarks of melanoma. Here, we report that the transcription factor Snail1 inhibits expression of the tumor suppressor CYLD in melanoma. As a direct consequence of CYLD repression, the protooncogene BCL-3 translocates into the nucleus and activates Cyclin D1 and N-cadherin promoters, resulting in proliferation and invasion of melanoma cells. Rescue of CYLD expression in melanoma cells reduced proliferation and invasion in vitro and tumor growth and metastasis in vivo. Analysis of a tissue microarray with primary melanomas from patients revealed an inverse correlation of Snail1 induction and loss of CYLD expression. Importantly, tumor thickness and progression-free and overall survival inversely correlated with CYLD expression. Our data suggest that Snail1-mediated suppression of CYLD plays a key role in melanoma malignancy.
Assuntos
Regulação Neoplásica da Expressão Gênica , Melanoma/patologia , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Animais , Proteína 3 do Linfoma de Células B , Western Blotting , Caderinas/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Ciclina D1/genética , Ciclina D1/metabolismo , Enzima Desubiquitinante CYLD , Regulação para Baixo , Feminino , Humanos , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos , Camundongos Nus , Invasividade Neoplásica , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/metabolismo , Transplante Heterólogo , Proteínas Supressoras de Tumor/metabolismoRESUMO
Brown adipose tissue (BAT) is a primary site of energy expenditure through thermogenesis, which is mediated by the uncoupling protein-1 (UCP-1) in mitochondria. Here, we show that protein kinase G (PKG) is essential for brown fat cell differentiation. Induction of adipogenic markers and fat storage was impaired in the absence of PKGI. Furthermore, PKGI mediated the ability of nitric oxide (NO) and guanosine 3',5'-monophosphate (cGMP) to induce mitochondrial biogenesis and increase the abundance of UCP-1. Mechanistically, we found that PKGI controlled insulin signaling in BAT by inhibiting the activity of RhoA and Rho-associated kinase (ROCK), thereby relieving the inhibitory effects of ROCK on insulin receptor substrate-1 and activating the downstream phosphoinositide 3-kinase-Akt cascade. Thus, PKGI links NO and cGMP signaling with the RhoA-ROCK and the insulin pathways, thereby controlling induction of adipogenic and thermogenic programs during brown fat cell differentiation.