RESUMO
Renal tissue plays a crucial function in maintaining homeostasis, making it vulnerable to xenobiotic toxicity. Pueraria montana has more beneficial potential against the various diseases and has long history used as a traditional Chinese medicine. But its effect against the renal cancer not scrutinize. The goal of this study is to see if Pueraria montana can protect rats from developing kidney tumors caused by diethylnitrosamine (DEN) and ferric nitrite (Fe-NTA). Wistar rats was selected for the current study and DEN (use as an inducer) and Fe-NTA (promoter) for induction the renal cancer. For 22 weeks, the rats were given orally Pueraria montana (12.5, 25, and 50 mg/kg) treatment. At regular intervals, the body weight and food intake were calculated. The rats were macroscopically evaluated for identification of cancer in the renal tissue. The renal tumor makers, renal parameters, antioxidant enzymes, phase I and II enzymes, inflammatory cytokines and mediators were estimated at end of the experimental study. Pueraria montana treated rats displayed the suppression of renal tumors, incidence of the tumors along with suppression of tumor percentage. Pueraria montana treated rats significantly (p < 0.001) increased body weight and suppressed the renal weight and food intake. It also reduced the level of renal tumor marker ornithine decarboxylase (ODC) and [3H] thymidine incorporation along with suppression of renal parameter such as uric acid, blood urea nitrogen (BUN), urea and creatinine. Pueraria montana treatment significantly (p < 0.001) altered the level of phase enzymes and antioxidant. Pueraria montana treatment significantly (p < 0.001) repressed the level of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), interleukin-6 (IL-6) and improved the level of interleukin-10 (IL-10). Pueraria montana treatment suppressed the level of prostaglandin (PGE2), cyclooxygenase-2 (COX-2), nuclear kappa B factor (NF-κB) and transforming growth factor beta 1 (TGF-ß1). Pueraria montana suppressed the inflammatory necrosis, size the bowman capsules in the renal histopathology. Pueraria montana exhibited the chemoprotective effect via dual mechanism such as suppression of inflammatory reaction and oxidative stress.
Assuntos
Neoplasias Renais , Pueraria , Animais , Antioxidantes/farmacologia , Biomarcadores Tumorais/metabolismo , Biomarcadores Tumorais/farmacologia , Peso Corporal , Creatinina/farmacologia , Ciclo-Oxigenase 2/metabolismo , Dietilnitrosamina/farmacologia , Compostos Férricos , Inflamação/tratamento farmacológico , Interleucina-10 , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Neoplasias Renais/induzido quimicamente , Neoplasias Renais/tratamento farmacológico , Neoplasias Renais/metabolismo , NF-kappa B/metabolismo , Ácido Nitrilotriacético/análogos & derivados , Nitritos/farmacologia , Ornitina Descarboxilase/metabolismo , Ornitina Descarboxilase/farmacologia , Estresse Oxidativo , Prostaglandinas , Prostaglandinas E/metabolismo , Prostaglandinas E/farmacologia , Pueraria/metabolismo , Ratos , Ratos Wistar , Timidina/metabolismo , Timidina/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Ureia , Ácido Úrico/farmacologia , Xenobióticos/farmacologiaRESUMO
The gut microbiota metabolizes drugs and alters their efficacy and toxicity. Diet alters drugs, the metabolism of the microbiota, and the host. However, whether diet-triggered metabolic changes in the microbiota can alter drug responses in the host has been largely unexplored. Here we show that dietary thymidine and serine enhance 5-fluoro 2'deoxyuridine (FUdR) toxicity in C. elegans through different microbial mechanisms. Thymidine promotes microbial conversion of the prodrug FUdR into toxic 5-fluorouridine-5'-monophosphate (FUMP), leading to enhanced host death associated with mitochondrial RNA and DNA depletion, and lethal activation of autophagy. By contrast, serine does not alter FUdR metabolism. Instead, serine alters E. coli's 1C-metabolism, reduces the provision of nucleotides to the host, and exacerbates DNA toxicity and host death without mitochondrial RNA or DNA depletion; moreover, autophagy promotes survival in this condition. This work implies that diet-microbe interactions can alter the host response to drugs without altering the drug or the host.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Floxuridina/toxicidade , Interações Alimento-Droga , Microbioma Gastrointestinal/efeitos dos fármacos , Serina/farmacologia , Animais , Caenorhabditis elegans/microbiologia , Caenorhabditis elegans/fisiologia , Suplementos Nutricionais , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Floxuridina/farmacocinética , Ácido Fólico/metabolismo , Microbioma Gastrointestinal/fisiologia , Timidina/análogos & derivados , Timidina/metabolismo , Timidina/farmacocinética , Timidina/farmacologia , Nucleotídeos de Uracila/metabolismo , Nucleotídeos de Uracila/farmacocinéticaRESUMO
Blood stasis (BS) is a complex syndrome with blood flow retardation or cessation. The Traditional Chinese Medicine, Curcumae rhizome (CR) and Sparganii rhizome (SR), showed promising effects on this disease, and especially effective when used in combination. However, the detailed influence of the TCMs on the BSS disturbed metabolic pathways was still unclear. In this study, a BS model was constructed in SD rat and the TCMs were used individually or in combination to assess the effects. As a result, combination of CR and SR led to the improvement in hemorheology parameters of up to 80% in the BS model. Further analyzing using metabolomics showed several metabolic pathways, including center carbon metabolism, amino acid metabolism, etc., recovered to the normal levels after treatment. Informatively, tyrosine and thymidine exhibited potential importance in the BSS and its treatment process. From these results, the metabolic profiles of BS and the SR-CR treatment were provided, which may helpful for better understanding the BSS mechanism and the development of more effective therapies.
Assuntos
Circulação Sanguínea/efeitos dos fármacos , Sangue/metabolismo , Curcuma/química , Medicamentos de Ervas Chinesas/farmacologia , Plerocercoide/química , Aminoácidos/metabolismo , Animais , Sangue/efeitos dos fármacos , Viscosidade Sanguínea/efeitos dos fármacos , Carbono/metabolismo , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/química , Masculino , Metabolômica/métodos , Microdiálise , Ratos Sprague-Dawley , Rizoma/química , Estresse Fisiológico/efeitos dos fármacos , Síndrome , Tempo de Trombina , Timidina/sangue , Timidina/metabolismoRESUMO
Comparative embryonic studies are the most effective way to discern phylogenetic changes. To gain insight into the constitution and evolution of mammalian somatosensory thalamic nuclei, we first studied how calbindin (CB) and parvalbumin (PV) immunoreactivities appear during embryonic development in the first-order relaying somatosensory nuclei, i.e., the ventral posteromedial (VPM) and posterolateral (VPL) nuclei, and their neighboring higher-order modulatory regions, including the ventromedial or ventrolateral nucleus, posterior, and the reticular nucleus. The results indicated that cell bodies that were immunoreactive for CB were found earlier (embryonic day 12 [E12]) in the dorsal thalamus than were cells positive for PV (E14), and the adult somatosensory thalamus was characterized by complementary CB and PV distributions with PV dominance in the first-order relaying nuclei and CB dominance in the higher-order regions. We then labeled proliferating cells with [(3) H]-thymidine from E11 to 19 and found that the onset of neurogenesis began later (E12) in the first-order relaying nuclei than in the higher-order regions (E11). Using double-labeling with [(3) H]-thymidine autoradiography and CB or PV immunohistochemistry, we found that CB neurons were born earlier (E11-12) than PV neurons (E12-13) in the studied areas. Thus, similar to auditory nuclei, the first and the higher-order somatosensory nuclei exhibited significant distinctions in CB/PV immunohistochemistry and birthdates during embryonic development. These data, combined with the results of a cladistic analysis of the thalamic somatosensory nuclei, are discussed from an evolutionary perspective of sensory nuclei.
Assuntos
Calbindinas/metabolismo , Neurogênese , Parvalbuminas/metabolismo , Núcleos Talâmicos/citologia , Núcleos Talâmicos/metabolismo , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Autorradiografia , Embrião de Mamíferos , Camundongos , Neurônios , Núcleos Talâmicos/embriologia , Núcleos Talâmicos/crescimento & desenvolvimento , Timidina/metabolismo , Trítio/metabolismoRESUMO
Nucleoside reverse transcriptase inhibitors (NRTIs)/nucleotide reverse transcriptase inhibitors are key components of combination antiretroviral therapy for HIV infection. First-generation NRTIs are associated with mitochondrial toxicity in patients, mainly due to inhibition of human DNA polymerase γ (hDNA polγ) that manifests as adverse events such as lipodystrophy, lactic acidosis, myopathy, cardiomyopathy, or nephropathy in patients. In chronic nonclinical studies in rodents and nonrodents, eukaryotic (host) mitochondrial toxicity manifests as some drug-specific toxicities similar to human toxicity. BMS-986001, a novel thymidine analog with minimal hDNA polγ inhibition, has demonstrated antiretroviral activity in early clinical studies. The primary toxicity of BMS-986001 in rats and monkeys is bone marrow dyserythropoiesis with associated decreases in red blood cell mass. Additionally, at high doses, severe platelet reductions accompanied by cutaneous petechiae began during weeks 8 and 11 in 3 of 60 monkeys in chronic toxicity studies. In a 6-month study, platelet reductions required euthanasia of the 2 affected monkeys (300 mg/kg/d) at week 14, but with dose reduction (200 mg/kg/d) remaining monkeys had no platelet changes. One affected monkey (200 mg/kg/d) in a 9-month study completed dosing and its platelet counts recovered during a 1-month recovery. Formation of platelet-bound immunoglobulin in the presence of BMS-986001, together with rapid and complete platelet recovery in the absence of BMS-986001, suggested that platelet decreases in monkeys may be immune mediated. No findings indicative of mitochondrial toxicity were observed in rats or monkeys given BMS-986001, suggesting an improved safety profile compared to marketed NRTI or tenofovir disoproxil fumarate.
Assuntos
Anemia Macrocítica/induzido quimicamente , Fármacos Anti-HIV/efeitos adversos , Drogas em Investigação/efeitos adversos , Púrpura Trombocitopênica/induzido quimicamente , Inibidores da Transcriptase Reversa/efeitos adversos , Timidina/análogos & derivados , Anemia Macrocítica/sangue , Anemia Macrocítica/metabolismo , Anemia Macrocítica/patologia , Animais , Fármacos Anti-HIV/administração & dosagem , Fármacos Anti-HIV/sangue , Fármacos Anti-HIV/metabolismo , Biotransformação , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Drogas em Investigação/administração & dosagem , Drogas em Investigação/metabolismo , Eritropoese/efeitos dos fármacos , Feminino , HIV-1/efeitos dos fármacos , HIV-1/crescimento & desenvolvimento , Meia-Vida , Macaca fascicularis , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Púrpura Trombocitopênica/imunologia , Púrpura Trombocitopênica/metabolismo , Púrpura Trombocitopênica/patologia , Distribuição Aleatória , Ratos Sprague-Dawley , Inibidores da Transcriptase Reversa/administração & dosagem , Inibidores da Transcriptase Reversa/sangue , Inibidores da Transcriptase Reversa/metabolismo , Análise de Sobrevida , Timidina/administração & dosagem , Timidina/efeitos adversos , Timidina/sangue , Timidina/metabolismo , Testes de Toxicidade Crônica , ToxicocinéticaRESUMO
This study aimed to investigate the mechanisms underlying the anti-proliferative effects of the ethanolic Cimicifuga racemosa extract BNO-1055 on prostate cells and evaluate its therapeutic potential. BNO-1055 dose-dependently attenuated cellular uptake and incorporation of thymidine and BrdU and significantly inhibited cell growth after long-time exposure. Similar results were obtained using saponin-enriched sub-fractions of BNO-1055. These inhibitory effects of BNO-1055 could be mimicked using pharmacological inhibitors and isoform-specific siRNAs targeting the equilibrative nucleoside transporters ENT1 and ENT2. Moreover, BNO-1055 attenuated the uptake of clinically relevant nucleoside analogs, e.g. the anti-cancer drugs gemcitabine and fludarabine. Consistent with inhibition of the salvage nucleoside uptake pathway BNO-1055 potentiated the cytotoxicity of the de novo nucleotide synthesis inhibitor 5-FU without significantly altering its uptake. Collectively, these data show for the first time that the anti-proliferative effects of BNO-1055 result from hindered nucleoside uptake due to impaired ENT activity and demonstrate the potential therapeutic use of BNO-1055 for modulation of nucleoside transport.
Assuntos
Antineoplásicos Fitogênicos/uso terapêutico , Proteínas de Transporte de Nucleosídeo Equilibrativas/metabolismo , Nucleosídeos/metabolismo , Fitoterapia , Extratos Vegetais/uso terapêutico , Próstata/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Antimetabólitos Antineoplásicos/farmacologia , Antimetabólitos Antineoplásicos/uso terapêutico , Antineoplásicos Fitogênicos/farmacologia , Bromodesoxiuridina/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Cimicifuga , Desoxicitidina/análogos & derivados , Desoxicitidina/metabolismo , Relação Dose-Resposta a Droga , Fluoruracila/farmacologia , Fluoruracila/uso terapêutico , Humanos , Masculino , Extratos Vegetais/farmacologia , Neoplasias da Próstata/metabolismo , RNA Interferente Pequeno/farmacologia , Saponinas/farmacologia , Saponinas/uso terapêutico , Timidina/metabolismo , Vidarabina/análogos & derivados , Vidarabina/metabolismo , GencitabinaRESUMO
UNLABELLED: The ornithine decarboxylase inhibitor α-difluoromethylornithine (DFMO) is a highly effective chemopreventive agent for colorectal cancer thought to act via polyamine depletion. However, in DFMO-treated patients, mucosal polyamine levels do not directly correlate with colorectal cancer risk. Untargeted metabolite profiling was used to broadly survey DFMO actions on colon cancer cell metabolism. We found that DFMO treatment of Apc(Min) intestinal tumors and human colorectal cancer cells is associated with reduced levels of folate-dependent metabolites, including S-adenosylmethionine (SAM), thymidine pools, and related pathway intermediates. We hypothesized that unrestrained SAM consumption/regeneration constitutes a futile DFMO-triggered cascade that can steal tetrahydrofolate from thymidylate synthase and thereby diminish thymidine pools. In accord with this hypothesis, DFMO treatment altered the folate cofactor balance and thymidine supplementation prevented DFMO-elicited cytostasis without restoring polyamine levels. These findings suggest that thymidine metabolite pool insufficiency is a fundamental mechanism of DFMO cytostatic activity. SIGNIFICANCE: A previously unappreciated metabolic linkage between polyamine and thymidine biosynthesis is revealed, based on the competing requirement of these pathways for a limited pool of tetrahydrofolate cofactor. This study identifies the fi rst shared mechanism for colorectal cancer chemoprevention and chemotherapy, suggesting a common metabolic target for both premalignant and malignant colon cells.
Assuntos
Neoplasias Colorretais/tratamento farmacológico , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Timidina/metabolismo , Timidina/farmacologia , Animais , Linhagem Celular Tumoral , Quimioprevenção , Neoplasias Colorretais/prevenção & controle , Células HT29 , Humanos , Camundongos , Inibidores da Ornitina Descarboxilase , S-Adenosilmetionina/metabolismoRESUMO
Aluminum (Al) has been considered as one of the most abundant elements and comprises nearly 8 % of the Earth's crust. Despite of its immense presence, studies regarding the molecular basis of its interaction with the physiological system are rather sparse. On the other hand, zinc (Zn), an essential micronutrient, has been regarded as the second most important metal for brain functioning. The objective of the present study was to investigate the protective potential of Zn, if any, during Al-induced detrimental effects on DNA, tritiated thymidine uptake as well as expression of stress marker genes and proteins in rat brain. Male Sprague-Dawley rats weighing 140-160 g were divided into four different groups viz.: normal control, Al treated (100 mg/kg b wt/day via oral gavage), Zn treated (227 mg/l in drinking water), and combined Al and Zn treated. All the treatments were carried out for a total duration of 8 weeks. Agarose gel electrophoresis revealed DNA laddering pattern and comets in the rat brain following Al treatment, which however, were attenuated upon Zn treatment. Further, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells, number of apoptotic brain cells, and uptake of tritiated thymidine were increased after Al treatment but were decreased upon Zn supplementation. Western blot and mRNA expressions of p53 and nuclear factor κB (NF-κB) were also found to be significantly elevated after Al treatment, which however, were reversed following Zn treatment. Hence, Zn shall prove to be an effective agent in mitigating the detrimental effects caused by Al in the rat brain.
Assuntos
Alumínio/toxicidade , Dano ao DNA , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Zinco/farmacologia , Animais , Apoptose/efeitos dos fármacos , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cérebro/efeitos dos fármacos , Cérebro/metabolismo , Ensaio Cometa , DNA/isolamento & purificação , Fragmentação do DNA/efeitos dos fármacos , Densitometria , Eletroforese em Gel de Ágar , Marcação In Situ das Extremidades Cortadas , Masculino , Microscopia de Fluorescência , NF-kappa B/metabolismo , Ratos , Ratos Sprague-Dawley , Timidina/metabolismo , Trítio/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Mesangial cell proliferation is correlated with the progression of renal failure. The purpose of this study was to determine whether a water extract of Poria cocos Wolf (WPC), a well-known medicinal plant, regulates rat mesangial cell proliferation in the presence of high glucose (HG). HG significantly accelerated [(3)H]-thymidine incorporation, which was inhibited by WPC (1-50 µg/mL) in a dose-dependent manner. Cell migration and fibronectin mRNA expression data also supported the anti-proliferative effect of WPC. Western blot analysis revealed that pretreatment with WPC decreased the expression of cyclins and cyclin-dependent kinases (CDKs) and promoted the expression of p21(waf1/cip1) and p27(kip1). WPC also suppressed HG-induced p38 mitogen-activated protein kinase (p38 MAPK) and extracellular-signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. Furthermore, WPC inhibited HG-induced production of dichlorofluorescein (DCF)-sensitive intracellular reactive oxygen species (ROS). In conclusion, HG promoted mesangial cell proliferation, and WPC inhibited this activity, at least in part, via induction of cell cycle arrest and activation of anti-oxidant properties. Taken together, these results suggest that P. cocos may be a potent regulator of HG-induced proliferation.
Assuntos
Proliferação de Células/efeitos dos fármacos , Glucose/efeitos adversos , Glucose/antagonistas & inibidores , Células Mesangiais/citologia , Extratos Vegetais/farmacologia , Poria , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Células Cultivadas , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Relação Dose-Resposta a Droga , Sistema de Sinalização das MAP Quinases , Masculino , Células Mesangiais/efeitos dos fármacos , Células Mesangiais/metabolismo , Fosforilação , Ratos , Espécies Reativas de Oxigênio/metabolismo , Timidina/metabolismo , Água , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Alkaline phosphatase (AP) is a ubiquitously expressed enzyme which can neutralize endotoxin as well as adenosine triphosphate (ATP), an endogenous danger signal released during brain injury. In this study we assessed a potential therapeutic role for AP in inhibiting neuroinflammation using three complementary approaches. METHODS: Mice were immunized to induce experimental autoimmune encephalomyelitis (EAE) and treated with AP for seven days during different phases of disease. In addition, serological assays to determine AP activity, endotoxin levels and endotoxin-reactive antibodies were performed in a cohort of multiple sclerosis (MS) patients and controls. Finally, the expression of AP and related enzymes CD39 and CD73 was investigated in brain tissue from MS patients and control subjects. RESULTS: AP administration during the priming phase, but not during later stages, of EAE significantly reduced neurological signs. This was accompanied by reduced proliferation of splenocytes to the immunogen, myelin oligodendrocyte glycoprotein peptide. In MS patients, AP activity and isoenzyme distribution were similar to controls. Although endotoxin-reactive IgM was reduced in primary-progressive MS patients, plasma endotoxin levels were not different between groups. Finally, unlike AP and CD73, CD39 was highly upregulated on microglia in white matter lesions of patients with MS. CONCLUSIONS: Our findings demonstrate that: 1) pre-symptomatic AP treatment reduces neurological signs of EAE; 2) MS patients do not have altered circulating levels of AP or endotoxin; and 3) the expression of the AP-like enzyme CD39 is increased on microglia in white matter lesions of MS patients.
Assuntos
Trifosfato de Adenosina/uso terapêutico , Fosfatase Alcalina/metabolismo , Encefalomielite Autoimune Experimental/tratamento farmacológico , Endotoxinas/metabolismo , Trifosfato de Adenosina/sangue , Adulto , Animais , Antígenos CD/metabolismo , Vasos Sanguíneos/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Endotoxinas/farmacologia , Ensaio de Imunoadsorção Enzimática , Feminino , Antígenos HLA-DR/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Esclerose Múltipla , Glicoproteína Mielina-Oligodendrócito/toxicidade , Fragmentos de Peptídeos/toxicidade , Mudanças Depois da Morte , Estatísticas não Paramétricas , Linfócitos T/efeitos dos fármacos , Timidina/metabolismo , Trítio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adulto JovemRESUMO
The effect of noncytotoxic doses of argemone oil (AO) and butter yellow (BY), the common adulterants in edible oil, on free radical generation and signaling pathway for cell proliferation in primary cells of gall bladder (GB) was undertaken. AO and BY showed no cytotoxicity at 0.1 µl/ml and 0.1 µg/ml concentration, respectively. AO caused significant increase in ROS after 30 min and RNS after 24 h in GB cells while no change was observed following BY treatment. Enhanced level of COX-2 was observed following AO (0.1 µl/ml) and BY (0.1 µg/ml) treatment to cells for 24 h. AO treatment caused phosphorylation of ErbB2, AKT, ERK, and JNK along with increased thymidine uptake indicating cell proliferation ability in GB cells. BY treatment also showed significant expression of these proteins with the exception of phosphorylated JNK. These results suggest that AO and BY have cell proliferative potential in GB cells following up-regulation of COX-2 and ErbB2; however, their downstream signaling molecules and free radical generation have differential response, indicating that the mechanism of proliferation is different for both compounds and may have relevance in gall bladder cancer.
Assuntos
Proliferação de Células , Vesícula Biliar/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases , Óleos de Plantas/toxicidade , Receptor ErbB-2/metabolismo , p-Dimetilaminoazobenzeno/toxicidade , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Meios de Cultura/metabolismo , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dimetil Sulfóxido/metabolismo , Relação Dose-Resposta a Droga , Vesícula Biliar/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Fosforilação , Cultura Primária de Células , Espécies Reativas de Nitrogênio/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor ErbB-2/genética , Timidina/metabolismo , Fatores de Tempo , Testes de Toxicidade/métodosRESUMO
The suppression of sprout growth is critical for the long-term storage of potato tubers. 1,4-Dimethylenapthlene (DMN) is a new class of sprout control agent but the metabolic mode of action for this compound has yet to be elucidated. Changes in transcriptional profiles of meristems isolated from potato tubers treated with the DMN were investigated using an Agilent 44 K 60-mer-oligo microarray. RNA was isolated from nondormant Russet Burbank meristems isolated from tubers treated with DMN for 3 days or activated charcoal as a control. RNA was used to develop probes that were hybridized against a microarray developed by the Potato Oligo Chip Initiative. Analysis of the array data was conducted in two stages: total array data was examined using a linear model and the software Limma and pathway analysis was conducted by linking the potato sequences to the Arabidopsis thaliana. DMN elicited a change in a number of transcripts associated with cold responses, water regulation, salt stress, and osmotic adjustment. DMN also resulted in a repression of cyclin or cyclin-like transcripts. DMN also resulted in a 50% decrease in thymidine incorporation suggesting a repression of the S phase of the cell cycle. Quantitative real-time polymerase chain reaction analysis demonstrated that DMN increased transcripts for the cell cycle inhibitors KRP1 and KRP2. We conclude the DMN results in alteration of genes associated with the maintenance of a G1/S phase block possibly through the induction of the cell cycle inhibitors KRP1 and KRP2.
Assuntos
Proteínas Inibidoras de Quinase Dependente de Ciclina/metabolismo , Naftalenos/farmacologia , Proteínas de Plantas/metabolismo , Inibidores de Proteínas Quinases/metabolismo , Solanum tuberosum/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Ciclo Celular/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Meristema/genética , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Proteínas de Plantas/genética , RNA de Plantas/genética , Reação em Cadeia da Polimerase em Tempo Real , Fase S , Solanum tuberosum/genética , Solanum tuberosum/crescimento & desenvolvimento , Timidina/metabolismo , Transcrição GênicaRESUMO
Chondrocytes from the lateral trochlear ridge of the distal femur taken from 1-day-old piglets were cultured in medium supplemented with 0, 7.8, 15.6, 31.2, and 62.5 µmol/L copper. Insulin-like growth factor-1 (IGF-1) and IGF-binding protein 3 (IGFBP-3) levels in culture medium were determined by radioimmunoassay. DNA synthesis in chondrocytes was measured by tritiated thymidine ((3)H-TdR) incorporation. Proliferation-promoting activity and incorporation of (3)H-TdR in chondrocytes were increased in all culture media supplemented with copper and 15% fetal calf serum (FCS). The contents of IGF-1 and IGFBP-3 were also enhanced significantly in culture media containing 15% FCS and supplemented with copper at 15.6, 31.2, and 62.5 µmol/L. The optimal copper concentration for promoting chondrocyte proliferation and autocrine secretion of IGF-1 and IGFBP-3 was 31.2 µmol/L.
Assuntos
Comunicação Autócrina/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Condrócitos/metabolismo , Sulfato de Cobre/farmacologia , Proteína 3 de Ligação a Fator de Crescimento Semelhante à Insulina/biossíntese , Fator de Crescimento Insulin-Like I/biossíntese , Animais , Animais Recém-Nascidos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Meios de Cultura , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , DNA/biossíntese , DNA/genética , Relação Dose-Resposta a Droga , Suínos , Timidina/metabolismoRESUMO
BACKGROUND: Several published studies indicate that the acyclic guanine nucleoside analogues possessing bis(1,2-hydroxymethyl) substituted cyclopropane rings mimicking the sugar moiety are potent inhibitors of replication of several herpes viruses. METHODS: Established synthetic methods and antiviral and cytostatic activity assays were used for the evaluation of new 1,2,4-triazole and purine acyclic nucleoside analogues. RESULTS: The synthesis of new types of acyclic nucleoside analogues which incorporate 1,2,4-triazole or purine moiety bound via flexible methylenic spacer to the bis(1,2-hydroxymethyl) cyclopropane ring. None of the new compounds showed pronounced antiviral activities at subtoxic concentrations on a broad panel of DNA and RNA viruses. Evaluation of their affinity for herpes simplex type 1 (HSV-1) and varicella-zoster virus-encoded thymidine kinases (VZV TK) also showed that none of the compounds was able to significantly inhibit 1 µM deoxythymidine phosphorylation by HSV-1 and VZV TK at 500 µM concentrations. The in vitro cytostatic activity evaluation results indicated a weak antiproliferative activity for all tested compounds. Only 6-pyrrolylpurine derivative bearing a carboxylic group substituted cyclopropane ring produced a rather slight inhibitory effect at higher micromolar concentrations on a breast carcinoma cell line (MCF-7) and no cytotoxic effect on human normal fibroblasts (WI 38). CONCLUSIONS: The lack of antiherpetic activity may be due to poor, if any, recognition of the compounds by virus-induced nucleoside kinases as an alternative substrate to become metabolically activated.
Assuntos
Antivirais/síntese química , Antivirais/farmacologia , Citostáticos/síntese química , Citostáticos/farmacologia , Nucleosídeos de Purina/química , Animais , Antivirais/química , Linhagem Celular Tumoral , Células Cultivadas , Citostáticos/química , Vírus de DNA/efeitos dos fármacos , Cães , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Herpesvirus Humano 1/enzimologia , Herpesvirus Humano 3/enzimologia , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Fosforilação , Nucleosídeos de Purina/farmacologia , Vírus de RNA/efeitos dos fármacos , Relação Estrutura-Atividade , Timidina/metabolismo , Timidina Quinase/antagonistas & inibidores , Triazóis/químicaRESUMO
Positron emission tomography (PET) imaging has become a useful tool for assessing early biologic response to cancer therapy and may be particularly useful in the development of new cancer therapeutics. RAF265, a novel B-Raf/vascular endothelial growth factor receptor-2 inhibitor, was evaluated in the preclinical setting for its ability to inhibit the uptake of PET tracers in the A375M(B-Raf(V600E)) human melanoma cell line. RAF265 inhibited 2-deoxy-2-[(18)F]fluoro-d-glucose (FDG) accumulation in cell culture at 28 hours in a dose-dependent manner. RAF265 also inhibited FDG accumulation in tumor xenografts after 1 day of drug treatment. This decrease persisted for the remaining 2 weeks of treatment. DNA microarray analysis of treated tumor xenografts revealed significantly decreased expression of genes regulating glucose and thymidine metabolism and revealed changes in apoptotic genes, suggesting that the imaging tracers FDG, 3-deoxy-3-[(18)F]fluorothymidine, and annexin V could serve as potential imaging biomarkers for RAF265 therapy monitoring. We concluded that RAF265 is highly efficacious in this xenograft model of human melanoma and decreases glucose metabolism as measured by DNA microarray analysis, cell culture assays, and small animal FDG PET scans as early as 1 day after treatment. Our results support the use of FDG PET in clinical trials with RAF265 to assess early tumor response. DNA microarray analysis and small animal PET studies may be used as complementary technologies in drug development. DNA microarray analysis allows for analysis of drug effects on multiple pathways linked to cancer and can suggest corresponding imaging tracers for further analysis as biomarkers of tumor response.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Perfilação da Expressão Gênica , Imidazóis/uso terapêutico , Leucemia Mieloide Aguda/tratamento farmacológico , Melanoma/tratamento farmacológico , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Piridinas/uso terapêutico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Avaliação Pré-Clínica de Medicamentos , Feminino , Fluordesoxiglucose F18 , Glucose/metabolismo , Humanos , Técnicas Imunoenzimáticas , Leucemia Mieloide Aguda/diagnóstico por imagem , Leucemia Mieloide Aguda/patologia , Melanoma/diagnóstico por imagem , Melanoma/patologia , Camundongos , Camundongos Nus , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/genética , Cintilografia , Compostos Radiofarmacêuticos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Timidina/metabolismo , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The present study evaluated the modulatory effects of zinc on 1,2-dimethylhydrazine (DMH)-induced ultrastructural changes in rat colon as well as on [(3)H]thymidine uptake and [(14)C]D-glucose metabolism. The rats were segregated into four groups: normal control, DMH treated, zinc treated, DMH + zinc treated. Initiation and induction of colon carcinogenesis was achieved through weekly subcutaneous injections of DMH (30 mg/kg body weight) for 8 and 16 weeks, respectively. Zinc was supplemented to rats at a dose level of 227 mg/L in drinking water, ad libitum for two different time durations of 8 and 16 weeks. The study revealed a significant decrease in zinc concentration in serum and colon following DMH treatment to rats, which upon zinc supplementation were recovered to near normal levels. A significant increase in in vitro [(3)H]thymidine uptake was observed following 16 weeks of DMH treatment. Further, a significant increase in the [(14)C]glucose turnover was observed following 8 and 16 weeks of DMH treatment. Simultaneous supplementation of zinc to DMH-treated rats for 16 weeks significantly decreased the uptake of [(3)H]thymidine and [(4)C]glucose when compared to DMH alone-treated rats. Changes in the ultrastructural architecture of colonic cells were evident following both treatment schedules of DMH; however, the changes were more distinguishable following 16 weeks of DMH treatment. The most obvious changes were seen in nuclear shape and disruption of cellular integrity, which upon zinc supplementation was appreciably improved. In conclusion, the study suggests positive beneficial effect of zinc against chemically induced colonic preneoplastic progression in rats.
Assuntos
1,2-Dimetilidrazina/toxicidade , Colo/efeitos dos fármacos , Neoplasias do Colo/ultraestrutura , Zinco/farmacologia , Animais , Colo/ultraestrutura , Neoplasias do Colo/induzido quimicamente , Neoplasias do Colo/prevenção & controle , Glucose/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Timidina/metabolismo , Zinco/sangueRESUMO
The mechanism of action of TNP-470 [O-(chloroacetyl-carbamoyl) fumagillol], which potently and selectively inhibits the proliferation of endothelial cells, is incompletely understood. Previous studies have established its binding protein and the most distal effector of its growth arrest activity as methionine aminopeptidase 2 (MetAP-2) and p21(WAF1/CIP1), respectively. However, the mechanistic steps between these two effectors have not been identified. We have found that addition of exogenous guanine and guanine-containing nucleosides to culture medium will completely reverse the cytostatic effect of TNP-470 on both cultured bovine aortic and mouse pulmonary endothelial cells. Western blotting showed that supplementation with exogenous guanosine reverses the induction of p21(WAF1/CIP1) by TNP-470. This "rescue" by guanine/guanosine was abolished when the guanine salvage pathway of nucleotide biosynthesis was inhibited with Immucillin H, suggesting that TNP-470 might reduce de novo guanine synthesis in endothelial cells. However, an analysis of inosine 5'-monophosphate dehydrogenase, the rate-limiting enzyme in de novo guanine synthesis and target of the antiangiogenic drug mycophenolic acid, showed no TNP-470-induced changes. Curiously, quantitation of cellular nucleotides confirmed that GTP levels were not reduced after TNP-470 treatment. Addition of guanosine at the start of G(1) phase causes a doubling in GTP levels that persists to the G(1)/S phase transition, where commitment to TNP-470 growth arrest occurs. Thus, guanine rescue involves an augmentation of cellular GTP beyond physiological levels rather than a restoration of a drug-induced GTP deficit. Determining the mechanism whereby this causes restoration of endothelial cell proliferation is an ongoing investigation.
Assuntos
Antibióticos Antineoplásicos/antagonistas & inibidores , Antibióticos Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Cicloexanos/antagonistas & inibidores , Cicloexanos/farmacologia , Células Endoteliais/efeitos dos fármacos , Nucleotídeos de Guanina/farmacologia , Guanina/farmacologia , Sesquiterpenos/antagonistas & inibidores , Sesquiterpenos/farmacologia , Trifosfato de Adenosina/biossíntese , Animais , Western Blotting , Bovinos , Ciclo Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Indução Enzimática/efeitos dos fármacos , Citometria de Fluxo , Imunofluorescência , Camundongos , Ácido Micofenólico/farmacologia , O-(Cloroacetilcarbamoil)fumagilol , Timidina/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/biossínteseRESUMO
Microorganisms play key roles in the cycles of carbon and nutrients in the ocean, and identifying the extent to which specific taxa contribute to these cycles will establish their ecological function. We examined the use of (33)P-phosphate to identify heterotrophic bacteria actively involved in the cycling of phosphate, an essential inorganic nutrient. Seawater from the sub-tropical North Atlantic Ocean was incubated with (33)P-phosphate and analysed by microautoradiography to determine the proportion and diversity of the bacterial community-assimilating phosphate. Complementary incubations using (3)H-leucine and (3)H-thymidine were also conducted. We found that a higher proportion of total heterotrophic bacterial cells in surface water samples assimilated phosphate compared with leucine or thymidine. Bacteria from all of the phylogenetic groups we identified by CARD-FISH were able to assimilate phosphate, although the abundances of cells within each group did not scale directly with the number found to assimilate phosphate. Furthermore, a significantly higher proportion of Alphaproteobacteria, Gammaproteobacteria and Cytophaga-like cells assimilated phosphate compared with leucine or thymidine. Our results suggest that a greater proportion of bacterial cells in surface waters are actively participating in the biogeochemical cycling of phosphorus, and possibly other elements, than is currently estimated through the use of (3)H-leucine or (3)H-thymidine.
Assuntos
Organismos Aquáticos/metabolismo , Bactérias/metabolismo , Fosfatos/metabolismo , Água do Mar/microbiologia , Poluentes Químicos da Água/metabolismo , Organismos Aquáticos/classificação , Oceano Atlântico , Bactérias/classificação , Fenômenos Ecológicos e Ambientais , Processos Heterotróficos , Leucina/análise , Leucina/metabolismo , Fosfatos/análise , Radioisótopos de Fósforo/análise , Água do Mar/química , Timidina/análise , Timidina/metabolismo , Poluentes Químicos da Água/análiseRESUMO
Established treatments for psoriasis are generally based on antiproliferative, anti-inflammatory, or differentiation-modifying activity, or a combination of these effects. New agents for the treatment of psoriasis could be identified by high-throughput screening (HTS) of large compound libraries using keratinocyte proliferation models. Although several new proliferation assays have been developed, the radioactive [(3)H]-thymidine incorporation assay is still considered to be the gold standard for the evaluation of keratinocyte proliferation in vitro. In this study, we compare a number of simple, and reliable, colorimetric (MTT, NRU, SRB, and CVS), and fluorimetric (CAM and AB) methods with the [(3)H]-thymidine incorporation assay for the measurement of keratinocyte proliferation in the exponential growth phase in 96-well formats. The concentrations that induced 50% growth inhibition (GI(50)) were determined by each assay for the established antipsoriatics, dithranol, and methotrexate. Strong correlations were observed between the percentage growth inhibitions determined by the radioactive and the colorimetric assays, with no significant differences (P > 0.05) between their GI(50) values. The colorimetric assays are thus suitable alternatives to the radioactive assay for quantifying keratinocyte growth inhibition. We have also validated the use of the HaCaT cell line as a representative of the hyperproliferative psoriatic epidermis, in the preclinical screening of experimental anti-psoriatic agents.
Assuntos
Proliferação de Células/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Psoríase/tratamento farmacológico , Timidina/metabolismo , Antralina/farmacologia , Anti-Infecciosos Locais/farmacologia , Caspase 3/metabolismo , Caspase 7/metabolismo , Linhagem Celular , Colorimetria , Fármacos Dermatológicos/farmacologia , Relação Dose-Resposta a Droga , Corantes Fluorescentes , Fluorometria , Humanos , Queratinócitos/efeitos dos fármacos , Metotrexato/farmacologia , Reprodutibilidade dos Testes , Sais de Tetrazólio , TiazóisRESUMO
Staphylococcus aureus (S. aureus) is one of the most common pathogens that causes infectious and foodborne diseases worldwide. Searching for drug and chemical compounds against this bacterium is still in demand. We found that grape seed extract (GSE), a natural food product rich in polyphenols, inhibited the dihydrofolate reductase activity and growth of S. aureus. In addition, the intracellular content of tetrahydrofolate (THF), the major folate species identified in S. aureus, was significantly decreased when GSE was present in medium. The GSE-induced growth inhibition was reversed by adding, THF, 5,10-methylenetetrahydrofolate or methionine to the medium. The differential rescuing effects elicited by thymidine and methionine indicated that GSE-induced perturbation in folate-mediated one-carbon metabolism has more profound impact on methionine cycle than on thymidine monophosphate (TMP) synthesis. Significantly reduced inflammatory responses and mortality were observed in zebrafish infected with S. aureus pre-incubated with GSE. We conclude that GSE might serve as an effective natural alternative for the control of food poisoning caused by S. aureus with proper safety measure.