RESUMO
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is the fourth most common cause of cancer related death. It is lethal in nearly all patients, due to an almost complete chemoresistance. Most if not all drugs that pass preclinical tests successfully, fail miserably in the patient. This raises the question whether traditional 2D cell culture is the correct tool for drug screening. The objective of this study is to develop a simple, high-throughput 3D model of human PDAC cell lines, and to explore mechanisms underlying the transition from 2D to 3D that might be responsible for chemoresistance. METHODS: Several established human PDAC and a KPC mouse cell lines were tested, whereby Panc-1 was studied in more detail. 3D spheroid formation was facilitated with methylcellulose. Spheroids were studied morphologically, electron microscopically and by qRT-PCR for selected matrix genes, related factors and miRNA. Metabolic studies were performed, and a panel of novel drugs was tested against gemcitabine. RESULTS: Comparing 3D to 2D cell culture, matrix proteins were significantly increased as were lumican, SNED1, DARP32, and miR-146a. Cell metabolism in 3D was shifted towards glycolysis. All drugs tested were less effective in 3D, except for allicin, MT100 and AX, which demonstrated effect. CONCLUSIONS: We developed a high-throughput 3D cell culture drug screening system for pancreatic cancer, which displays a strongly increased chemoresistance. Features associated to the 3D cell model are increased expression of matrix proteins and miRNA as well as stromal markers such as PPP1R1B and SNED1. This is supporting the concept of cell adhesion mediated drug resistance.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/genética , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas da Matriz Extracelular/genética , Neoplasias Pancreáticas/genética , Fenótipo , Esferoides Celulares/efeitos dos fármacos , Animais , Antineoplásicos/toxicidade , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Ensaios de Seleção de Medicamentos Antitumorais , Metabolismo Energético , Proteínas da Matriz Extracelular/metabolismo , Humanos , Ácido Láctico/metabolismo , Camundongos , Neoplasias Pancreáticas/metabolismo , Células Tumorais CultivadasRESUMO
The effects of thyroxine and its related compounds on the benzodiazepine-insensitive γ-aminobutyric acid type A (GABA(A)) receptors were studied. Thyroxine at micromolar concentrations potentiated the (3)H-Ro15-4513 binding to rat brain membranes in-vitro in the thalamus, striatum, cortex and hippocampus, but not in cerebellum. In the thalamus, the rank order of potency was the following: 3,3',5,5'-tetraiodothyroacetic acid (TETRAC)>L-thyroxine>3,5-diiodo-l-thyronine (3,5-T2). TETRAC induced a slight potentiation of flumazenil binding to diazepam-sensitive GABA(A) receptors in the thalamus and striatum while no effect was found in cortex and hippocampus. Consequently, we examined whether these compounds could exert their modulatory effect on the currents mediated by benzodiazepine-insensitive GABA(A) receptors. The diazepam-insensitive GABA(A) receptor-mediated currents were recorded from acutely isolated rat ventrobasal thalamic neurons by applying low concentrations of 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP). TETRAC and thyroxine at low µM concentrations potentiated the THIP-evoked currents, although 3,5-T2 had no effect on the THIP-induced currents. Ethanol had no effect on the enhancing effects of TETRAC. TETRAC itself evoked GABA(A) receptor-mediated currents at high concentrations beyond 30 µM. Although the effects of TETRAC and thyroxine were observed at non-physiological concentrations of hormones, the present results might lead to new lead structures with specificity to diazepam-insensitive GABA(A) receptor subtypes.
Assuntos
Diazepam/farmacologia , Moduladores GABAérgicos/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Tiroxina/farmacologia , Marcadores de Afinidade , Animais , Azidas/metabolismo , Benzodiazepinas/metabolismo , Encéfalo/efeitos dos fármacos , Depressores do Sistema Nervoso Central/farmacologia , Fenômenos Eletrofisiológicos , Etanol/farmacologia , Isoxazóis , Masculino , Membranas/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Hormônios Tireóideos/sangue , Hormônios Tireóideos/farmacologia , Tiroxina/análogos & derivados , Tiroxina/sangueRESUMO
BACKGROUND: Thyroid hormones (TH) regulate cholesterol metabolism but their use as lipid-lowering drugs is restricted due to negative cardiac effects. TH mimetic compounds modulating TH receptor ß (THRß) have been designed as potential drugs, reducing serum cholesterol levels while avoiding apparent deleterious cardiac effects. OBJECTIVE: Using ApoE deficient mice, we examined whether KB3495, a TH mimetic compound, reduces atherosclerosis and if there is a synergistic effect with atorvastatin. The effect of KB3495 was investigated after 10 and 25 weeks. RESULTS: KB3495 treatment reduced atherosclerotic plaque formation in aorta and decreased the cholesteryl ester (CE) content by 57%. Treatment with KB3495 was also associated with a reduction of macrophage content in the atherosclerotic plaques and reduced serum levels of IL-1ß, TNFalpha, IL-6, Interferon γ, MCP-1 and M-CSF. Serum lipoprotein analysis showed no change in total cholesterol levels in ApoB-containing lipoproteins. KB3495 alone increased fecal BA excretion by 90%. The excretion of neutral sterols increased in all groups, with the largest increase in the combination group (350%). After 25 weeks, the animals treated with KB3495 showed 50% lower CE levels in the skin and even further reductions were observed in the combination group where the CE levels were reduced by almost 95% as compared to controls. CONCLUSION: KB3495 treatment reduced atherosclerosis independently of total cholesterol levels in ApoB-containing lipoproteins likely by stimulation of sterol excretion from the body and by inhibition of the inflammatory response.
Assuntos
Anticolesterolemiantes/farmacologia , Aterosclerose/tratamento farmacológico , Ácidos Heptanoicos/farmacologia , Placa Aterosclerótica/prevenção & controle , Pirróis/farmacologia , Animais , Anticolesterolemiantes/síntese química , Apolipoproteínas B/metabolismo , Apolipoproteínas E/deficiência , Apolipoproteínas E/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Atorvastatina , Ácidos e Sais Biliares/metabolismo , Transporte Biológico/efeitos dos fármacos , Citocinas/biossíntese , Citocinas/metabolismo , Modelos Animais de Doenças , Sinergismo Farmacológico , Fezes/química , Masculino , Camundongos , Camundongos Knockout , Mimetismo Molecular , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Hormônios Tireóideos/químicaRESUMO
Coordinated regulation of bile acid biosynthesis, the predominant pathway for hepatic cholesterol catabolism, is mediated by few key nuclear receptors including the orphan receptors liver receptor homolog 1 (LRH-1), hepatocyte nuclear factor 4alpha (HNF4alpha), small heterodimer partner (SHP), and the bile acid receptor FXR (farnesoid X receptor). Activation of FXR initiates a feedback regulatory loop via induction of SHP, which suppresses LRH-1- and HNF4alpha-dependent expression of cholesterol 7alpha hydroxylase (CYP7A1) and sterol 12alpha hydroxylase (CYP8B1), the two major pathway enzymes. Here we dissect the transcriptional network governing bile acid biosynthesis in human liver by identifying GPS2, a stoichiometric subunit of a conserved corepressor complex, as a differential coregulator of CYP7A1 and CYP8B1 expression. Direct interactions of GPS2 with SHP, LRH-1, HNF4alpha, and FXR indicate alternative coregulator recruitment strategies to cause differential transcriptional outcomes. In addition, species-specific differences in the regulation of bile acid biosynthesis were uncovered by identifying human CYP8B1 as a direct FXR target gene, which has implications for therapeutic approaches in bile acid-related human disorders.
Assuntos
Ácidos e Sais Biliares/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Transcrição Gênica , Carcinoma Hepatocelular , Linhagem Celular Tumoral , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Regulação Enzimológica da Expressão Gênica , Humanos , Neoplasias Hepáticas , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Esteroide 12-alfa-Hidroxilase/genética , Esteroide 12-alfa-Hidroxilase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In this review, estrogenic effects in depression, anxiety, and neurodegenerative disorders are summarized. Moreover, preclinical findings from in vitro and animal models are discussed. There is a correlation between decreased estrogen levels (e.g., premenstrually, during the postpartum period, and perimenopausally) and increased anxiety and depressive symptoms. Several studies show beneficial effects of estrogen treatment in women with anxiety and depressive symptoms. Recent data indicate that the estrogen receptor (ER) beta appears to be a major mediator of estrogenic effects in depression and anxiety. Additionally, both preclinical and clinical findings suggest that activation of estrogen receptors have an important role in neuroprotective and neurodegenerative processes in the mammalian central nervous system (CNS).