RESUMO
Primary human hepatocytes (hHeps) are still gold standard to perform human drug metabolism studies, but their availability is limited by donor organ scarcity. Therefore, hepatoma cell lines are widely used as alternatives, although their phases I and II drug-metabolizing activities are substantially lower compared with hHeps. The major advantage of these cell lines is immediate availability, standardized culture conditions and unlimited life span. Therefore, the aim of this study was to investigate the drug-metabolizing profile of five human hepatoma cell lines (HepG2, Hep3B, HCC-T, HCC-M and Huh-7) over a culture period of 10 passages. Fluorescent-based assays for seven different cytochrome P450 (CYP) isoforms and seven different phase II enzymes were performed and compared with enzymatic activities of hHeps. CYP activities were much lower in the cell lines (5-15% of hHeps), whereas phase II enzyme activities that are involved in buffering oxidative stress (e.g., Glutathione-S-transferase) reached levels comparable with hHeps. Furthermore, phases I and II enzyme activities in hepatoma cell lines vary strongly during culture time. Interestingly, the most constant results were obtained with Huh-7 cells. Huh-7 cells as well as HCC-T cells exhibited a drug-metabolizing profile closest to hHeps between passages two and four. Toxicity studies with Diclofenac, Paracetamol and Verapamil in both cell lines show dose-response characteristics and EC(50) values similar to hHeps. Therefore, we propose that due to the more consistent results throughout the passages, Huh-7 could be an alternative system to the limitedly available hHeps and frequently used HepG2 cell line in the study of drug metabolism.
Assuntos
Carcinoma Hepatocelular/enzimologia , Hepatócitos/enzimologia , Neoplasias Hepáticas/enzimologia , Preparações Farmacêuticas/metabolismo , Acetaminofen/metabolismo , Acetaminofen/toxicidade , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Sistema Enzimático do Citocromo P-450/metabolismo , Diclofenaco/metabolismo , Diclofenaco/toxicidade , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Hepatócitos/metabolismo , Humanos , Isoenzimas , Neoplasias Hepáticas/metabolismo , Estresse Oxidativo , Verapamil/metabolismo , Verapamil/toxicidadeRESUMO
PURPOSE: Recent reports discuss the altered bone homeostasis in cigarette smokers, being a risk factor for osteoporosis and negatively influencing fracture healing. Cigarette smoke is known to induce oxidative stress in the body via an increased production of reactive oxygen species (ROS). These increases in ROS are thought to damage the bone-forming osteoblasts. Naturally occurring polyphenols contained in green tea extract (GTE), e.g., catechins, are known to have anti-oxidative properties. Therefore, the aim of this study was to investigate whether GTE and especially catechins protect primary human osteoblasts from cigarette smoke-induced damage and to identify the underlying mechanisms. METHODS: Primary human osteoblasts were isolated from patients' femur heads. Cigarette smoke medium (CSM) was obtained using a gas-washing bottle and standardized by its optical density (OD(320)) at λ = 320 nm. ROS formation was measured using 2'7'dichlorofluorescein diacetate, and osteoblasts' viability was detected by resazurin conversion. RESULTS: Co-, pre-, and post-incubation with GTE and catechins significantly reduced ROS formation and thus improved the viability of CSM-treated osteoblasts. Besides GTE's direct radical scavenging properties, pre-incubation with both GTE and catechins protected osteoblasts from CSM-induced damage. Inhibition of the anti-oxidative enzyme HO-1 significantly reduced the protective effect of GTE and catechins emphasizing the key role of this enzyme in GTE anti-oxidative effect. CONCLUSIONS: Our data suggest possible beneficial effects on bone homeostasis, fracture healing, and bone mineral density following a GTE-rich diet or supplementation.
Assuntos
Camellia sinensis , Catequina/farmacologia , Osteoblastos/efeitos dos fármacos , Fumar/efeitos adversos , Densidade Óssea , Relação Dose-Resposta a Droga , Consolidação da Fratura , Heme Oxigenase-1/metabolismo , Humanos , Osteoporose , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismoRESUMO
Recent publications suggested that monocytes might be an attractive cell type to transdifferentiate into various cellular phenotypes. Aim was, therefore, to evaluate the potential of blood monocytes to transdifferentiate into osteoblasts. Monocytes isolated from peripheral blood were subjected to two previously published treatments to obtain unique, multipotent cell fractions, named programmable cells of monocytic origin (PCMOs) and monocyte-derived mesenchymal progenitor cells (MOMPs). Subsequently, MOMPs and PCMOs were treated with osteogenic differentiation medium (including either vitamin D or dexamethasone) for 14 days. Regarding a variety of surface markers, no differences between MOMPs, PCMOs, and primary monocytes could be detected. The treatment with osteogenic medium neither resulted in loss of hematopoietic markers nor in adoption of mesenchymal phenotype in all cell types. No significant effect was observed regarding the expression of osteogenic transcription factors, bone-related genes, or production of mineralized matrix. Osteogenic medium resulted in activation of monocytes and appearance of osteoclasts. In conclusion, none of the investigated monocyte cell types showed any transdifferentiation characteristics under the tested circumstances. Based on our data, we rather see an activation and maturation of monocytes towards macrophages and osteoclasts.
Assuntos
Transdiferenciação Celular , Monócitos/fisiologia , Osteoblastos/citologia , Fosfatase Alcalina/metabolismo , Células Cultivadas , Humanos , Macrófagos/citologia , Células-Tronco Mesenquimais , Células-Tronco Multipotentes , Osteoclastos/citologia , Osteogênese/fisiologiaRESUMO
Targeting the baculoviral inhibitor of apoptosis proteins repeat (BIR) 3 of X-linked inhibitor of apoptosis proteins (XIAP) represents an innovative strategy for the design of chemosensitizers. Acylated flavonol monorhamnosides (AFMR) from Eriobotrya japonica Lindl. (Rosaceae) were virtually predicted as ligands of the XIAP BIR3 domain by using a previously generated pharmacophore model. From the methanol leaf extract of E. japonica an enriched mixture of AFMR was obtained showing chemosensitizing potential in combination with etoposide in XIAP-overexpressing Jurkat cells. The HPLC-SPE-NMR hyphenated technique facilitated the structure elucidation of three known and two new natural AFMR. The main constituent and virtual hit, kaempferol-3-O-α-l-(2â³,4â³-di-E-p-coumaroyl)-rhamnoside (3) was isolated from the enriched fraction. Applying a fluorescence polarization based binding assay, 3 was identified as XIAP BIR3 ligand with a dose-dependent affinity (IC50 10.4 µM). Further, 3 induced apoptosis in XIAP-overexpressing Jurkat cells and activated caspase-9 in combination with etoposide. Docking experiments revealed a major impact of the coumaric acid and sugar moieties of 3 on XIAP BIR3 binding, which was experimentally confirmed. To conclude, this study elucidates 3 as natural, small-molecular weight XIAP BIR3 inhibitor using a combination of in silico and HPLC-SPE-NMR hyphenated techniques.
Assuntos
Eriobotrya/química , Flavonoides/química , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/antagonistas & inibidores , Sítios de Ligação , Linhagem Celular Tumoral , Simulação por Computador , Avaliação Pré-Clínica de Medicamentos , Flavonoides/isolamento & purificação , Flavonoides/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Folhas de Planta/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Although significant progress has been made in the field of orthotopic liver transplantation, cell-based therapies seem to be a promising alternative to whole-organ transplantation. The reasons are manifold but organ shortage is the main cause for this approach. However, many problems such as the question which cell type should be used or which application site is best for transplantation have been raised. In addition, some clinicians have had success by cultivating liver cells in bioreactors for temporary life support. Besides answering the question which cell type, which injection site or even which culture form should be used for liver support recent international harmonization of legal requirements is needed to be addressed by clinicians, scientists and companies dealing with cellular therapies. We here briefly summarize the possible cell types used to partially or temporarily correct liver diseases, the most recent development of bioreactor technology and important regulatory issues.
Assuntos
Hepatócitos/transplante , Hepatopatias/terapia , Falência Hepática/terapia , Fígado Artificial , Fígado/citologia , Reatores Biológicos , Técnicas de Cultura de Células , Criança , Pré-Escolar , Desenho de Equipamento , Feminino , Humanos , Lactente , Consentimento Livre e Esclarecido/legislação & jurisprudência , Masculino , Pessoa de Meia-IdadeRESUMO
Smokers frequently suffer from impaired fracture healing often due to poor bone quality and stability. Cigarette smoking harms bone cells and their homeostasis by increased formation of reactive oxygen species (ROS). The aim of this study was to investigate whether Quercetin, a naturally occurring antioxidant, can protect osteoblasts from the toxic effects of smoking. Human osteoblasts exposed to cigarette smoke medium (CSM) rapidly produced ROS and their viability decreased concentration- and time-dependently. Co-, pre- and postincubation with Quercetin dose-dependently improved their viability. Quercetin increased the expression of the anti-oxidative enzymes heme-oxygenase- (HO-) 1 and superoxide-dismutase- (SOD-) 1. Inhibiting HO-1 activity abolished the protective effect of Quercetin. Our results demonstrate that CSM damages human osteoblasts by accumulation of ROS. Quercetin can diminish this damage by scavenging the radicals and by upregulating the expression of HO-1 and SOD-1. Thus, a dietary supplementation with Quercetin could improve bone matter, stability and even fracture healing in smokers.
Assuntos
Heme Oxigenase-1/metabolismo , Osteoblastos/efeitos dos fármacos , Quercetina/farmacologia , Fumar/efeitos adversos , Superóxido Dismutase/metabolismo , Antioxidantes/metabolismo , Sobrevivência Celular , Meios de Cultura/metabolismo , Ativação Enzimática , Inibidores Enzimáticos , Humanos , Osteoblastos/enzimologia , Cultura Primária de Células , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase-1 , Fatores de Tempo , Regulação para CimaRESUMO
Extracts of the traditional medicinal herb Andrographis paniculata and its main constituents are described in the literature as showing anticancer activity. The aim of this study was to isolate the main constituents of a commercially available phytotherapeutic preparation of A. paniculata and to determine their chemosensitizing potential in a leukemia cell line. Chromatographic separation steps resulted in the isolation of the diterpenes andrographolide (1) and 14-deoxy-11,12-didehydroandrographolide (3) and the diterpene glucoside neoandrographolide (2). The combination of these constituents with suboptimal concentrations of etoposide revealed compound 2 as chemosensitizer in S-Jurkat and X chromosome-linked inhibitor of apoptosis protein (XIAP)-overexpressing Jurkat cells, a model for chemoresistance.
Assuntos
Andrographis/química , Antineoplásicos Fitogênicos/farmacologia , Diterpenos/farmacologia , Glucosídeos/farmacologia , Extratos Vegetais/farmacologia , Tetra-Hidronaftalenos/farmacologia , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/isolamento & purificação , Fracionamento Químico , Diterpenos/química , Diterpenos/isolamento & purificação , Glucosídeos/química , Glucosídeos/isolamento & purificação , Humanos , Células Jurkat , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Tetra-Hidronaftalenos/química , Tetra-Hidronaftalenos/isolamento & purificaçãoRESUMO
BACKGROUND: Despite extensive research, the underlying pathological mechanisms of osteoporosis are not completely understood. Recent studies have indicated a distinct role for the IFN-ß/STAT1 pathway in bone metabolism. An inhibitory effect of IFN-ß on osteoclastogenesis has been detected and STAT1/2 has been shown to influence osteoblastic bone metabolism. So far, no data concerning the IFN-ß/STAT1 pathways in osteoblasts and osteoclasts from osteoporotic and non-osteoporotic patients are available. The aim of the study was to analyze these pathways in both cell types. METHODS: Osteoblasts were isolated from the femoral heads of 12 osteoporotic and 11 non-osteoporotic patients and monocytes were differentiated into osteoclasts. After the differentiation period, cells were stimulated once with 20 and 100 ng/mL IFN-ß for 4 days. Viability, activity, bone metabolism-related genes, and the proteins Fra1, SOCS1, STAT1, p-STAT1, and TRAF6 were analyzed. RESULTS: Viability, activity, and gene expressions were not affected by stimulating the osteoblasts. However, in osteoporotic osteoclasts, which display a significantly higher basal osteoclastic activity, the stimulation with IFN-ß lead to significant inhibition. Further, an increased STAT1 activation was detected in both cell types with no significant differences between the groups. Regarding the phosphorylation of STAT1, no significant influence was detected in osteoblasts but the IFN-ß stimulation led to a significant increase of p-STAT1 in osteoclasts of both groups. CONCLUSIONS: IFN-ß is a principal mediator in the pathogenesis of osteoporosis by inhibiting osteoclasts and inducing and activating STAT1. Our results also confirm this in cells from osteoporotic and non-osteoporotic patients. Strong inhibitory effects on the osteoclastogenesis of osteoporotic osteoclasts were detectable. Nevertheless, osteoblast activity was not negatively affected by IFN-ß stimulation. These results may contribute to a better understanding of the underlying pathological signaling pathways of osteoporosis.
Assuntos
Interferon beta/farmacologia , Osteoporose/metabolismo , Fator de Transcrição STAT1/metabolismo , Transdução de Sinais , Células Cultivadas , Humanos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteogênese , Osteoporose/etiologia , Osteoporose/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição STAT1/genética , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismoRESUMO
Proximal femur fracture, a typical fracture of the elderly, is often associated with morbidity, reduced quality of life, impaired physical function and increased mortality. There exists evidence that responses of the hematopoietic microenvironment to fractures change with age. Therefore, we investigated oxidative stress markers and oxidative stress-related MAPK activation in granulocytes from the young and the elderly with and without fractured long bones. Lipid peroxidation levels were increased in the elderly controls and patients. Aged granulocytes were more sensitive towards oxidative stress induced damage than young granulocytes. This might be due to the basally increased expression of SOD-1 in the elderly, which was not further induced by fractures, as observed in young patients. This might be caused by an altered MAPK activation. In aged granulocytes basal p38 and JNK activities were increased and basal ERK1/2 activity was decreased. Following fracture, JNK activity decreased, while ERK1/2 and p38 activities increased in both age groups. Control experiments with HL60 cells revealed that the observed p38 activation depends strongly on age. Summarizing, we observed age-dependent changes in the oxidative stress response system of granulocytes after fractures, for example, altered MAPK activation and SOD-1 expression. This makes aged granulocytes vulnerable to the stress stimuli of the fracture and following surgery.
Assuntos
Granulócitos/patologia , Fraturas do Quadril/patologia , Fraturas do Quadril/fisiopatologia , Estresse Oxidativo , Regeneração , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Sobrevivência Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Granulócitos/efeitos dos fármacos , Granulócitos/enzimologia , Células HEK293 , Células HL-60 , Fraturas do Quadril/enzimologia , Humanos , Peróxido de Hidrogênio/toxicidade , Peroxidação de Lipídeos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo/efeitos dos fármacos , Regeneração/efeitos dos fármacos , Soro/metabolismo , Superóxido Dismutase/metabolismo , Adulto Jovem , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The therapeutic value of adipose-derived mesenchymal stem cells (Ad-MSCs) for bone regeneration is critically discussed. A possible reason for reduced osteogenic potential may be an age-related deterioration of the Ad-MSCs. In long term in vitro culture, epigenomic changes in DNA methylation are known to cause gene silencing, affecting stem cell growth as well as the differentiation potential. In this study, we observed an age-related decline in proliferation of primary human Ad-MSCs. Decreased Nanog, Oct4 and Lin28A and increased Sox2 gene-expression was accompanied by an impaired osteogenic differentiation potential of Ad-MSCs isolated from old donors (>60 a) as compared to Ad-MSCs isolated from younger donors (<45 a). 5-hydroxymethylcytosine (5 hmC) and 5-methylcytonsine (5 mC) distribution as well as TET gene expression were evaluated to assess the evidence of active DNA demethylation. We observed a decrease of 5 hmC in Ad-MSCs from older donors. Incubation of these cells with 5-Azacytidine induced proliferation and improved the osteogenic differentiation potential in these cells. The increase in AP activity and matrix mineralization was associated with an increased presence of 5 hmC as well as with an increased TET2 and TET3 gene expression. Our data show, for the first time, a decrease of DNA hydroxymethylation in Ad-MSCs which correlates with donor-age and that treatment with 5-Azacytidine provides an approach which could be used to rejuvenate Ad-MSCs from aged donors.
Assuntos
Azacitidina/farmacologia , Diferenciação Celular , Metilação de DNA/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Tecido Adiposo Branco/citologia , Envelhecimento , Proliferação de Células , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Metilases de Modificação do DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Dioxigenases/metabolismo , Epigênese Genética , Expressão Gênica , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Osteocalcina/genética , Osteocalcina/metabolismo , Osteogênese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fator de Transcrição Sp7 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The use of isolated human liver cells in research and development has gained increasing interest during the past years. The possible application may vary between elucidation of new biochemical pathways in liver diseases, drug development, safety issues, and new therapeutic strategies up to direct clinical translation for liver support. However, the isolation of human liver cells requires a well-developed logistic network among surgeons, biologists, and technicians to obtain a high quality of cells. Our laboratories have been involved in various applications of human liver cells and we have long-lasting experiences in human liver cell isolation and their application in R&D. We here summarize the present protocol of our laboratories for cell isolation from normal resected liver tissue, the most common tissue available. In addition, we discuss the necessary network in the clinic and quality controls to maintain human liver cells in culture and the effect of 3D extracellular matrix in cultured cells which results in preservation of hepatocyte epithelial polarity in the form of bile canaliculi and repression of epithelial to mesenchymal transitions occurring in 2D cultures.
Assuntos
Separação Celular/métodos , Hepatócitos/citologia , Cultura Primária de Células/métodos , Cultura Primária de Células/normas , Células Cultivadas , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Humanos , Controle de QualidadeRESUMO
AIMS: Mitochondria not only generate and modulate bioenergy but also serve as biosensors for oxidative stress, and eventually become effector organelles for cell viability. Therefore, the implications of mitochondrial (dys)function in the development of multiple organ failure are profound. We investigated whether a mutation in the ATPase subunit-8 gene affects the course of endotoxemic acute liver failure. MAIN METHODS: C57BL/6J (ATP8 wild type) and C57BL/6J-mt(FVB/N) (ATP8 mutant) mice were challenged with d-galactosamine (GalN) and Escherichia coli lipopolysaccharide (LPS) for induction of acute liver failure, and studied 6 h thereafter. Control mice received physiological saline only. Analysis included in vivo fluorescence microscopy of hepatic microcirculation and levels of hepatocellular apoptosis, hepatic adenosine nucleotides and oxidative stress. Additionally, survival rates were assessed. KEY FINDINGS: Induction of endotoxemic liver failure provoked marked liver damage, which was coexistent with a drop of total adenosine nucleotide levels and increased oxidative stress. Of interest, oxidative stress was higher in the GalN/LPS challenged ATP8 mutants compared to wild types. Concomitantly, adenosine triphosphate (ATP) levels in livers of mice carrying the ATP8 mutation remained higher than those in wild type mice. As net result, ATP8 mutants showed lower transaminase release and a tendency to better survival rate upon GalN/LPS exposure compared to wild types. SIGNIFICANCE: Our findings demonstrate that mutation in the ATPase subunit-8 partially protects mice against endotoxemic stress, most probably due to better hepatic energy status despite elevated oxidative stress. Thus, modulating mitochondrial function to preserve bioenergetic status may be an effective strategy to protect against sepsis-induced multiorgan dysfunction.
Assuntos
Endotoxemia/genética , Genes Mitocondriais/genética , Falência Hepática Aguda/genética , Fígado/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , Animais , Apoptose/fisiologia , Western Blotting , Modelos Animais de Doenças , Endotoxemia/metabolismo , Endotoxemia/patologia , Endotoxinas/farmacologia , Metabolismo Energético/genética , Metabolismo Energético/fisiologia , Genes Mitocondriais/fisiologia , Fígado/patologia , Falência Hepática Aguda/metabolismo , Falência Hepática Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL/genética , Camundongos Mutantes , Microscopia de Fluorescência , Mutação , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologiaRESUMO
Spongistatin 1, a marine experimental substance with chemotherapeutic potential, induces apoptosis and inhibits clonogenic survival of MCF-7 cells. Regarding the apoptotic signaling pathways of spongistatin 1, we present two major facts. Firstly, spongistatin 1-induced cell death, mainly caspase-independent, involves the proapoptotic proteins apoptosis-inducing factor and endonuclease G. Both proteins translocate from mitochondria to the nucleus and contribute to spongistatin 1-mediated apoptosis as shown via gene silencing. Secondly, spongistatin 1 acts as a tubulin depolymerizing agent and is able to free the proapoptotic Bcl-2 family member Bim from its sequestration both by the microtubular complex and by the antiapoptotic protein Mcl-1. Silencing of Bim by small interfering RNA leads to a diminished translocation of apoptosis-inducing factor and endonuclease G to the nucleus and subsequently reduces apoptosis rate. Thus, we identified Bim as an important factor upstream of mitochondria executing a central role in the caspase-independent apoptotic signaling pathway induced by spongistatin 1. Taken together, spongistatin 1 is both a valuable tool for the characterization of apoptotic pathways and a promising experimental anticancer drug.