RESUMO
The effective concentration of a drug in the blood, i.e. the concentration of a free drug in the blood, is influenced by the strength of drug binding onto plasma proteins. Besides its efficacy, these interactions subsequently influence the liberation, absorption, distribution, metabolism, excretion, and toxicological properties of the drug. It is important to not only determine the binding strength and stoichiometry, but also the binding site of a drug on the plasma protein molecule, because the co-administration of drugs with the same binding site can affect the above-mentioned concentration and as a result the pharmacological behavior of the drugs and lead to side effects caused by the change in free drug concentration, its toxicity. In this study, the binding characteristics of six drugs with human serum albumin, the most abundant protein in human plasma, were determined by capillary electrophoresis-frontal analysis, and the obtained values of binding parameters were compared with the literature data. The effect of several drugs and site markers on the binding of l-tryptophan and lidocaine to human serum albumin was investigated in subsequent displacement studies which thus demonstrated the usability of capillary electrophoresis as an automated high-throughput screening method for drug-protein binding studies.
Assuntos
Clorpropamida/análise , Diclofenaco/análise , Flurbiprofeno/análise , Ibuprofeno/análise , Fenilbutazona/análise , Tolbutamida/análise , Sítios de Ligação/efeitos dos fármacos , Clorpropamida/farmacologia , Diclofenaco/farmacologia , Eletroforese Capilar , Flurbiprofeno/farmacologia , Humanos , Ibuprofeno/farmacologia , Lidocaína/antagonistas & inibidores , Lidocaína/química , Fenilbutazona/farmacologia , Albumina Sérica Humana/química , Tolbutamida/farmacologia , Triptofano/antagonistas & inibidores , Triptofano/químicaRESUMO
Binding strength of the anti-diabetic drugs chlorpropamide (CPM) and tolbutamide (TBM) with model protein bovine serum albumin (BSA) shows strong modulation in presence of colloidal gold nanoparticles (AuNP). Intrinsic tryptophan fluorescence of both the native BSA and BSA-AuNP conjugate quenched in presence of the drugs. Stern-Volmer quenching constant (KSV) of CPM binding to BSA-AuNP conjugate at different temperatures is almost twice (6.76~14.76 × 103 M-1) than the corresponding values in native BSA (3.21~5.72 × 103 M-1). However, the calculated KSV values with TBM show certain degree of reduction in presence of AuNP (6.46× 103 M-1), while comparing with native BSA (8.83 × 103 M-1). The binding mode of CPM towards BSA-AuNP conjugate is mainly through hydrophobic forces; whereas, TBM binding is identified to be Van der Waal's and hydrogen bonding type of interaction. Fluorescence lifetime analysis confirms static type of quenching for the intrinsic tryptophan fluorescence of BSA as well as BSA-AuNP conjugate with addition of CPM and TBM at different concentrations. The α-helical content in the secondary structure of BSA is decreased to 48.32% and 45. 28% in presence of AuNP, when the concentration of CPM is 0.08 mM and 0.16 mM in comparison with that of native protein (50.13%). On the other hand, the intensity of sugar induced advanced glycated end (AGE) product fluorescence is decreased by 55% and 80% at 0.13 nM and 0.68 nM AuNP, respectively. Change in the binding strength of the drugs with transport protein and reduced AGE product formation in presence of AuNP could lead to a major development in the field of nanomedicine and associated drug delivery techniques. Graphical Abstract Modulated drug binding ability and AGE product formation of serum proteins in presence of AuNP.
Assuntos
Clorpropamida/farmacologia , Produtos Finais de Glicação Avançada/antagonistas & inibidores , Hipoglicemiantes/farmacologia , Soroalbumina Bovina/química , Tolbutamida/farmacologia , Adsorção , Animais , Sítios de Ligação/efeitos dos fármacos , Bovinos , Clorpropamida/química , Coloides/química , Produtos Finais de Glicação Avançada/metabolismo , Ouro/química , Ouro/farmacologia , Interações Hidrofóbicas e Hidrofílicas , Hipoglicemiantes/química , Nanopartículas Metálicas/química , Tamanho da Partícula , Soroalbumina Bovina/antagonistas & inibidores , Soroalbumina Bovina/metabolismo , Espectrometria de Fluorescência , Propriedades de Superfície , Temperatura , Tolbutamida/químicaRESUMO
Canagliflozin, used to treat type 2 diabetes mellitus (T2DM), is commonly co-administered with sulfonylureas. The objective of the present study was to evaluate the possible inhibitory effect of sulfonylureas and non-steroidal anti-inflammatory drugs (NSAIDs) on canagliflozin metabolism in vitro. Three sulfonylurea derivatives were evaluated as inhibitors: chlorpropamide, glimepiride and gliclazide. Two other NSAIDs were used as positive control inhibitors: niflumic acid and diclofenac. The rate of formation of canagliflozin metabolites was determined by HPLC analysis of in vitro incubations of canagliflozin as a substrate with and without inhibitors, using human liver microsomes (HLMs). Among sulfonylureas, glimepiride showed the most potent inhibitory effect against canagliflozin M7 metabolite formation, with an IC50 value of 88 µm, compared to chlorpropamide and gliclazide with IC50 values of more than 500 µm. Diclofenac inhibited M5 metabolite formation more than M7, with IC50 values of 32 µm for M5 and 80 µm for M7. Niflumic acid showed no inhibition activity against M5 formation, but had relatively selective inhibitory potency against M7 formation, which is catalysed by UGT1A9, with an IC50 value of 1.9 µm and an inhibition constant value of 0.8 µm. A clinical pharmacokinetic interaction between canagliflozin and sulfonylureas is unlikely. However, a possible clinically important drug interaction between niflumic acid and canagliflozin has been identified.
Assuntos
Canagliflozina/metabolismo , Clorpropamida/farmacologia , Diclofenaco/farmacologia , Gliclazida/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Ácido Niflúmico/farmacologia , Compostos de Sulfonilureia/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Canagliflozina/farmacocinética , Relação Dose-Resposta a Droga , Interações Medicamentosas , Humanos , Hipoglicemiantes/farmacologiaRESUMO
Theca cell-selective Pten mutation (tPtenMT) in mice resulted in increases in PDK1 and Akt phosphorylation, indicating an over-activation of PI3K signaling in the ovaries. These mice displayed elevated androgen levels, ovary enlargement, antral follicle accumulation, early fertility loss and increased expression of Lhcgr and genes that are crucial to androgenesis. These abnormalities were partially reversed by treatments of PI3K or Akt inhibitor. LH actions in Pten deficient theca cells were potentiated. The phosphorylation of Foxo1 was increased, while the binding of Foxo1 to forkhead response elements in the Lhcgr promoter was reduced in tPtenMT theca cells, implying a mechanism by which PI3K/Akt-induced upregulation of Lhcgr in theca cells might be mediated by reducing the inhibitory effect of Foxo1 on the Lhcgr promoter. The phenotype of tPtenMT females is reminiscent of human PCOS and suggests that dysregulated PI3K cascade in theca cells may be involved in certain types of PCOS pathogenesis.
Assuntos
Androgênios/metabolismo , Deleção de Genes , Ovário/fisiopatologia , PTEN Fosfo-Hidrolase/genética , Células Tecais/metabolismo , Envelhecimento/metabolismo , Animais , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Feminino , Fertilidade , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Hormônio Luteinizante/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Modelos Biológicos , Ovário/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Receptores do LH/genética , Receptores do LH/metabolismo , Esteroides/biossíntese , Testosterona/sangue , Células Tecais/efeitos dos fármacosRESUMO
Phosphorylated Ser473-Akt (p-Ser473-Akt) is extensively studied as a correlate for the activity of Akt, which plays an important role in mouse oogenesis and preimplantation embryogenesis. However, little progress has been made about its effect on the mouse zygotic genome activation (ZGA) of 2-cell stage in mouse preimplantation embryos. In this study, we confirmed its localization in the pronuclei of 1-cell embryos and found that p-Ser473-Akt acquired prominent nucleus localization in 2-cell embryos physiologically. Akt specific inhibitors API-2 and MK2206 could inhibit the development of mouse preimplantation embryos in vitro, and induce 2-cell arrest at certain concentrations. 2-cell embryos exposed to 2.0 µmol/L API-2 or 30 µmol/L MK2206 displayed attenuated immunofluorescence intensity of p-Ser473-Akt in the nucleus. Simultaneously, qRT-PCR results revealed that 2.0 µmol/L API-2 treatment significantly downregulated the mRNA pattern of MuERV-L and eIF-1A, two marker genes of ZGA, suggesting a defect in ZGA compared with that of control group. Collectively, our work demonstrated the nuclear localization of p-Ser473-Akt during major ZGA, and Akt specific inhibitors API-2 and MK2206 which led to 2-cell arrest inhibited p-Ser473-Akt from translocating into the nucleus of 2-cell embryos with defective ZGA as well, implying p-Ser473-Akt may be a potential player in the major ZGA of 2-cell mouse embryos.
Assuntos
Blastocisto/metabolismo , Núcleo Celular/metabolismo , Desenvolvimento Embrionário , Proteínas Proto-Oncogênicas c-akt/metabolismo , Zigoto/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Animais , Blastocisto/citologia , Blastocisto/efeitos dos fármacos , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Técnicas de Cultura Embrionária , Fator de Iniciação 1 em Eucariotos/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Genoma/genética , Compostos Heterocíclicos com 3 Anéis/farmacologia , Masculino , Camundongos , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina/metabolismo , Zigoto/citologia , Zigoto/efeitos dos fármacosRESUMO
Myocardial infarction, a prevalent cardiovascular disease, is associated with cardiomyocyte cell death, and eventually heart failure. Cardiac tissue engineering has provided hopes for alternative treatment options, and high-fidelity tissue models for drug discovery. The signal transduction mechanisms relayed in response to mechanoelectrical (physical) stimulation or biochemical stimulation (hormones, cytokines, or drugs) in engineered heart tissues (EHTs) are poorly understood. In this study, an EHT model was used to elucidate the signaling mechanisms involved when insulin was applied in the presence of electrical stimulation, a stimulus that mimics functional heart tissue environment in vitro. EHTs were insulin treated, electrically stimulated, or applied in combination (insulin and electrical stimulation). Electrical excitability parameters (excitation threshold and maximum capture rate) were measured. Protein kinase B (AKT) and phosphatidylinositol-3-kinase (PI3K) phosphorylation revealed that insulin and electrical stimulation relayed electrical excitability through two separate signaling cascades, while there was a negative crosstalk between sustained activation of AKT and PI3K.
Assuntos
Fenômenos Eletrofisiológicos , Coração/fisiologia , Fosfatidilinositol 3-Quinase/metabolismo , Engenharia Tecidual/métodos , Animais , Animais Recém-Nascidos , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Cromonas/farmacologia , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Insulina/farmacologia , Modelos Biológicos , Morfolinas/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos Sprague-DawleyRESUMO
OBJECTIVES: Cooling following cardiac arrest can improve survival significantly. However, delays in achieving target temperature may decrease the overall benefits of cooling. Here, we test whether lipid emulsion, a clinically approved drug reported to exert cardioprotection, can rescue heart contractility in the setting of delayed cooling in stunned mouse cardiomyocytes. DESIGN: Cell culture study. SETTING: Academic research laboratory. SUBJECTS: Cardiomyocytes isolated from 1- to 2-day-old C57BL6 mice. INTERVENTIONS: Cardiomyocytes were exposed to 30 minutes of ischemia followed by 90 minutes of reperfusion and 10 minutes of isoproterenol with nine interventions: 1) no additional treatment; 2) intraischemic cooling at 32 °C initiated 10 minutes prior to reperfusion; 3) delayed cooling started 20 minutes after reperfusion; 4) lipid emulsion + delayed cooling; 5) lipid emulsion (0.25%) administered at reperfusion; 6) lipid emulsion + intraischemic cooling; 7) delayed lipid emulsion; 8) lipid emulsion + delayed cooling + Akt inhibitor (API-2, 10 µM); and 9) lipid emulsion + delayed cooling + Erk inhibitor (U0126, 10 µM). Inhibitors were given to cells 1 hour prior to ischemia. MEASUREMENTS AND MAIN RESULTS: Contractility was recorded by real-time phase-contrast imaging and analyzed with pulse image velocimetry in MATLAB (Mathworks, Natick, MA). Ischemia diminished cell contraction. The cardioprotective effect of cooling was diminished when delayed but was rescued by lipid emulsion. Further, lipid emulsion on its own improved recovery of the contractility to a greater extent as intraischemic cooling. However, cotreatment of lipid emulsion and intraischemic cooling did not further improve the recovery compared to either treatment alone. Furthermore, Akt and Erk inhibitors blocked lipid emulsion-induced protection. CONCLUSIONS: Lipid emulsion improved contractility and rescued contractility in the context of delayed cooling. This protective effect required Akt and Erk signaling. Lipid emulsion might serve as a treatment or adjunct to cooling in ameliorating myocardial ischemia/reperfusion injury.
Assuntos
Butadienos/farmacologia , Cardiotônicos/farmacologia , Clorpropamida/análogos & derivados , Hipotermia Induzida/métodos , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miócitos Cardíacos/patologia , Nitrilas/farmacologia , Animais , Clorpropamida/farmacologia , Modelos Animais de Doenças , Isquemia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Contração Muscular/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Fatores de TempoRESUMO
BACKGROUND AND PURPOSE: Cellular radioresistance is a major impediment to effective radiotherapy. Here, we demonstrated that long-term exposure to fractionated radiation conferred acquired radioresistance to tumor cells due to AKT-mediated enhanced aerobic glycolysis. MATERIAL AND METHODS: Two human tumor cell lines with acquired radioresistance were established by long-term exposure to fractionated radiation with 0.5 Gy of X-rays. Glucose uptake was inhibited using 2-deoxy-D-glucose, a non-metabolizable glucose analog. Aerobic glycolysis was assessed by measuring lactate concentrations. Cells were then used for assays of ROS generation, survival, and cell death as assessed by annexin V staining. RESULTS: Enhanced aerobic glycolysis was shown by increased glucose transporter Glut1 expression and a high lactate production rate in acquired radioresistant cells compared with parental cells. Inhibiting the AKT pathway using the AKT inhibitor API-2 abrogated these phenomena. Moreover, we found that inhibiting glycolysis with 2-deoxy-D-glucose suppressed acquired tumor cell radioresistance. CONCLUSIONS: Long-term fractionated radiation confers acquired radioresistance to tumor cells by AKT-mediated alterations in their glucose metabolic pathway. Thus, tumor cell metabolic pathway is an attractive target to eliminate radioresistant cells and improve radiotherapy efficacy.
Assuntos
Neoplasias/metabolismo , Neoplasias/radioterapia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Aerobiose , Morte Celular/efeitos dos fármacos , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Desoxiglucose/farmacologia , Glucose/metabolismo , Transportador de Glucose Tipo 1/biossíntese , Glicólise , Células HeLa , Células Hep G2 , Humanos , Lactatos/metabolismo , Neoplasias/enzimologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Tolerância a Radiação/fisiologia , Tolerância a Radiação/efeitos da radiação , Espécies Reativas de Oxigênio/metabolismoRESUMO
The Wntß-catenin and PI3K-AKT-FOXO3a pathways have a central role in cancer. AKT phosporylates FOXO3a, relocating it from the cell nucleus to the cytoplasm, an effect that is reversed by PI3K and AKT inhibitors. Simultaneous hyperactivation of the Wntß-catenin pathway and inhibition of PI3K-AKT signaling promote nuclear accumulation of ß-catenin and FOXO3a, respectively, promoting cell scattering and metastasis by regulating a defined set of target genes. Indeed, the anti-tumoral AKT inhibitor API-2 promotes nuclear FOXO3a accumulation and metastasis of cells with high nuclear ß-catenin content. Nuclear ß-catenin confers resistance to the FOXO3a-mediated apoptosis induced by PI3K and AKT inhibitors in patient-derived primary cultures and in corresponding xenograft tumors in mice. This resistance is reversed by XAV-939, an inhibitor of Wntß-catenin signaling. In the presence of high nuclear ß-catenin content, activation of FOXO3a by PI3K or AKT inhibitors makes it behave as a metastasis inductor rather than a proapoptotic tumor suppressor. We show that it is possible to evaluate the ß-catenin status of patients' carcinomas and the response of patient-derived cells to target-directed drugs that accumulate FOXO3a in the nucleus before deciding on a course of treatment. We propose that this evaluation could be essential to the provision of a safer and more effective personalized treatment.
Assuntos
Neoplasias do Colo/patologia , Fatores de Transcrição Forkhead/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , beta Catenina/fisiologia , Animais , Apoptose , Linhagem Celular Tumoral , Núcleo Celular/química , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Doxiciclina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Feminino , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/análise , Humanos , Camundongos , Camundongos SCID , Metástase Neoplásica , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Via de Sinalização Wnt , beta Catenina/análiseRESUMO
There is an urgent need for the development of novel therapies to treat pancreatic cancer, which is among the most lethal of all cancers. KRAS-activating mutations, which are found in more than 90% of pancreatic adenocarcinomas, drive tumor dependency on the Ras/MAPK and Akt signaling pathways. Radiation is currently being explored as a component of the standard treatment regimen for pancreatic cancer. This study's purpose was to test the hypothesis that MAP kinase kinase (MEK or MAP2K) inhibitors will offer clear therapeutic benefit when integrated into radiotherapy treatment regimens for treatment of this disease. We explored the activation of the mitogen-activated protein kinase (MAPK) and Akt pathways in response to radiation in multiple pancreatic tumor cell lines. Small molecule inhibitors of MEK (PD0325901) and Akt (API-2) were subsequently evaluated for their radiosensitizing potential alone and in combination. In vivo efficacy was tested in subcutaneous MIA-PaCa2 xenografts. Phosphorylated levels of extracellular signal-regulated kinase (ERK)-1/2 and Akt were found to increase in response to radiation treatment in our pancreatic tumor cell line panel. MEK inhibitor-induced radiosensitization was observed in vitro and in vivo. The further addition of an Akt inhibitor to the MEK inhibitor/radiation regimen resulted in enhanced therapeutic gain as determined by increased radiosensitization and tumor cell death. In conclusion, MEK inhibition results in growth arrest, apoptosis, and radiosensitization of multiple preclinical pancreatic tumor models, and the effects can be enhanced by combination with an Akt inhibitor. These results provide rationale for further testing of a treatment regimen in pancreatic cancer that combines MEK inhibition with radiation, optimally in conjunction with Akt inhibition.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias Pancreáticas/terapia , Inibidores de Fosfoinositídeo-3 Quinase , Radiossensibilizantes/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Benzamidas/farmacologia , Benzamidas/uso terapêutico , Linhagem Celular Tumoral , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Clorpropamida/uso terapêutico , Terapia Combinada , Difenilamina/análogos & derivados , Difenilamina/farmacologia , Difenilamina/uso terapêutico , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/efeitos da radiação , Inibidores Enzimáticos/farmacologia , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/radioterapia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Radiossensibilizantes/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Albumin in the glomerular filtrate is normally retrieved by concerted efforts of clathrin, LDL-type receptor megalin- and clathrin-associated sorting proteins. In glomerular diseases, albumin overload triggers a proapoptotic and inflammatory response contributing to tubulointerstitial fibrosis and tubular atrophy. The relationship between albumin overload-induced proximal tubule injury and albumin endocytosis remains to be discovered. We investigated presence of a possible overlap between endocytosis and cell survival. We showed a novel interaction between prosurvival protein, protein kinase B (PKB/Akt), and adaptor protein, disabled 2 (Dab2), with coimmunoprecipitation. Further delineation of this interaction by GST pull-down experiments utilizing different Dab2 constructs identified proline-rich domain as the interacting partner. Expression of Dab2 and PKB/Akt was downregulated at high concentrations of albumin associated with apoptosis. We then examined the physiological relevance of this interaction with functional studies. Overexpression of PKB/Akt increased albumin uptake in human proximal tubule cells. Conversely, inhibition of PKB/Akt with a nonselective Akt/PKB signaling inhibitor-2 and a dominant negative construct of PKB/Akt resulted in a decrease in albumin uptake. Inhibition of Dab2 by silencing RNA abolished PKB/Akt-induced albumin uptake demonstrating the physiological importance of this novel interaction. We concluded that PKB/Akt is part of an endocytic machinery and it mediates albumin uptake through its interaction with Dab2. The role that PKB/Akt plays in the endocytic cascade may dictate its decreased expression in proteinuric states in an attempt to limit albumin endocytosis that may tilt the balance between cell survival and apoptosis toward cell death.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Albuminas/fisiologia , Endocitose/fisiologia , Túbulos Renais Proximais/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Proteínas Reguladoras de Apoptose , Linhagem Celular , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Regulação para Baixo , Endocitose/efeitos dos fármacos , Inativação Gênica , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Supressoras de TumorRESUMO
Inhibition of PKB (protein kinase B) activity using a highly selective PKB inhibitor resulted in inhibition of cell cycle progression only if cells were in early G1 phase at the time of addition of the inhibitor, as demonstrated by time-lapse cinematography. Addition of the inhibitor during mitosis up to 2 h after mitosis resulted in arrest of the cells in early G1 phase, as deduced from the expression of cyclins D and A and incorporation of thymidine. After 24 h of cell cycle arrest, cells expressed the cleaved caspase-3, a central mediator of apoptosis. These results demonstrate that PKB activity in early G1 phase is required to prevent the induction of apoptosis. Using antibodies, it was demonstrated that active PKB translocates to the nucleus during early G1 phase, while an even distribution of PKB was observed through cytoplasm and nucleus during the end of G1 phase.
Assuntos
Caspase 3/metabolismo , Clorpropamida/análogos & derivados , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Células CHO , Caspase 3/genética , Clorpropamida/farmacologia , Cricetinae , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Ciclinas/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Mitose/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Timidina/metabolismo , Imagem com Lapso de TempoRESUMO
Both insulin and the cell death-inducing DNA fragmentation factor-α-like effector (CIDE) family play important roles in apoptosis and lipid droplet formation. Previously, we reported that CIDEA and CIDEC are differentially regulated by insulin and contribute separately to insulin-induced anti-apoptosis and lipid droplet formation in human adipocytes. However, the upstream signals of CIDE proteins remain unclear. Here, we investigated the signaling molecules involved in insulin regulation of CIDEA and CIDEC expression. The phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and PI-103 blocked both insulin-induced downregulation of CIDEA and upregulation of CIDEC. The Akt inhibitor API-2 and the c-Jun N-terminal kinase (JNK) inhibitor SP600125 selectively inhibited insulin regulation of CIDEA and CIDEC expression, respectively, whereas the MAPK/ERK kinase inhibitor U0126 and the p38 inhibitor SB203580 did not. Small interfering RNA-mediated depletion of Akt1/2 prevented insulin-induced downregulation of CIDEA and inhibition of apoptosis. Depletion of JNK2, but not JNK1, inhibited insulin-induced upregulation of CIDEC and lipid droplet enlargement. Furthermore, insulin increased both Akt and JNK phosphorylation, which was abrogated by the PI3K inhibitors. These results suggest that insulin regulates CIDEA and CIDEC expression via PI3K, and it regulates expression of each protein via Akt1/2- and JNK2-dependent pathways, respectively, in human adipocytes.
Assuntos
Adipócitos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Regulação da Expressão Gênica , Insulina , Obesidade/metabolismo , Proteínas/metabolismo , Transdução de Sinais , Adipócitos/citologia , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Fragmentação do DNA/efeitos dos fármacos , Regulação para Baixo , Feminino , Furanos/farmacologia , Inativação Gênica/efeitos dos fármacos , Humanos , Insulina/metabolismo , Insulina/farmacologia , Proteína Quinase 9 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 9 Ativada por Mitógeno/genética , Proteína Quinase 9 Ativada por Mitógeno/metabolismo , Obesidade/genética , Obesidade/patologia , Obesidade/fisiopatologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/farmacologia , Regulação para CimaRESUMO
PURPOSE: Radioresistance is a major cause of treatment failure of radiotherapy (RT) in human cancer. We have recently revealed that acquired radioresistance of tumor cells induced by fractionated radiation is attributable to cyclin D1 overexpression as a consequence of the downregulation of GSK3ß-dependent cyclin D1 proteolysis mediated by a constitutively activated serine-threonine kinase, AKT. This prompted us to hypothesize that targeting the AKT/GSK3ß/cyclin D1 pathway may improve fractionated RT by suppressing acquired radioresistance of tumor cells. METHODS AND MATERIALS: Two human tumor cell lines with acquired radioresistance were exposed to X-rays after incubation with either an AKT inhibitor, AKT/PKB signaling inhibitor-2 (API-2), or a Cdk4 inhibitor (Cdk4-I). Cells were then subjected to immunoblotting, clonogenic survival assay, cell growth analysis, and cell death analysis with TUNEL and annexin V staining. In vivo radiosensitivity was assessed by growth of human tumors xenografted into nude mice. RESULTS: Treatment with API-2 resulted in downregulation of cyclin D1 expression in cells with acquired radioresistance. Cellular radioresistance disappeared completely both in vitro and in vivo with accompanying apoptosis when treated with API-2. Furthermore, inhibition of cyclin D1/Cdk4 by Cdk4-I was sufficient for abolishing radioresistance. Treatment with either API-2 or Cdk4-I was also effective in suppressing resistance to cis-platinum (II)-diamine-dichloride in the cells with acquired radioresistance. Interestingly, the radiosensitizing effect of API-2 was canceled by overexpression of cyclin D1 whereas Cdk4-I was still able to sensitize cells with cyclin D1 overexpression. CONCLUSION: Cyclin D1/Cdk4 is a critical target of the AKT survival signaling pathway responsible for tumor radioresistance. Targeting the AKT/GSK3ß/cyclin D1/Cdk4 pathway would provide a novel approach to improve fractionated RT and would have an impact on tumor eradication in combination with chemotherapy.
Assuntos
Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Tolerância a Radiação/efeitos dos fármacos , Radiossensibilizantes/farmacologia , Animais , Apoptose , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Cisplatino/farmacologia , Ciclina D1/antagonistas & inibidores , Fracionamento da Dose de Radiação , Regulação para Baixo , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Células HeLa , Células Hep G2 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/metabolismo , Tolerância a Radiação/fisiologia , Tolerância a Radiação/efeitos da radiação , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
INTRODUCTION AND OBJECTIVE: It has been suggested that type 2 diabetes is an inflammatory response manifestation. The main drugs used to treat type 2 diabetes are sulphonylureas and biguanides. The aim of this study was to demonstrate the modulatory effects of oral hypoglycemic drugs (chlorpropamide and metformin) on lymphocyte proliferation in vitro and ex vivo. METHODS: Peripheral blood mononuclear cells were isolated from human blood by gradient centrifugation. T-lymphocytes were stimulated by phytohemagglutinin (PHA) and oral hypoglycemic drugs. RESULTS: In both in vitro and ex vivo experiments, there was a reduction in cell proliferation after treatment with oral hypoglycemic drugs. When both drugs were used in combination, a high level of cytotoxicity was observed, which made analysis of immunomodulatory effects unfeasible. DISCUSSION AND CONCLUSION: We demonstrated that diabetes itself may reduce cell proliferation significantly when stimulated by PHA, which may indicate that diabetic patients have difficulties in promoting an efficient inflammatory response. Moreover, the use of oral hypoglycemic drugs may aggravate this situation.
INTRODUÇÃO E OBJETIVOS: Tem sido sugerido que o diabetes mellitus tipo 2 (DM2) é uma manifestação da resposta inflamatória. As principais drogas utilizadas no tratamento do DM2 são as sulfonilureias e as biguanidas. O objetivo deste trabalho é demonstrar os efeitos moduladores na proliferação de linfócitos causada pelos hipoglicemiantes orais (clorpropamida e metformina), in vitro e ex vivo. MÉTODOS: Células mononucleares de sangue periférico foram isoladas de seres humanos por gradiente de centrifugação. Os linfócitos T foram estimulados com fito-hemaglutinina (PHA) e hipoglicemiantes. RESULTADOS: Nos experimentos in vitro e ex vivo, mostramos a redução da proliferação celular quando do tratamento com drogas hipoglicemiantes orais. Quando as drogas foram utilizadas em combinação, foi observado alto grau de citotoxicidade, tornando inviável a análise do efeito imunomodulador. DISCUSSÃO E CONCLUSÃO: Mostramos que o diabetes, por si, pode reduzir significativamente a proliferação celular quando estimulada por PHA, o que pode indicar que o paciente diabético tem dificuldade em promover a eficiente resposta inflamatória e que o uso de hipoglicemiantes pode piorar esta situação.
Assuntos
Humanos , Clorpropamida/farmacologia , Fatores Imunológicos/farmacologia , Imunomodulação , Metformina/farmacologiaRESUMO
Drug resistance is a central challenge of cancer therapy that ultimately leads to treatment failure. In this study, we characterized a mechanism of drug resistance that arises to AZD6244, an established mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) 1/2 inhibitor currently being evaluated in cancer clinical trials. AZD6244 enhanced the expression of transcription factor FOXO3a, which suppressed cancer cell proliferation. In AZD6244-resistant cancer cells, we observed the impaired nuclear localization of FOXO3a, reduced FOXO3a-mediated transcriptional activity, and decreased the expression of FOXO3a target gene Bim after cell treatment with AZD6244. Resistant cells could be sensitized by phosphoinositide 3-kinase (PI3K)/AKT inhibitors, which are known to enhance FOXO3a nuclear translocation. Our findings define FOXO3a as candidate marker to predict the clinical efficacy of AZD6244. Furthermore, they suggest a mechanism of resistance to MEK inhibitors that may arise in the clinic yet can be overcome by cotreatment with PI3K/AKT inhibitors.
Assuntos
Benzimidazóis/farmacologia , Fatores de Transcrição Forkhead/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Cromonas/farmacologia , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Proteína Forkhead Box O3 , Células HCT116 , Células HT29 , Humanos , MAP Quinase Quinase 1/antagonistas & inibidores , MAP Quinase Quinase 1/metabolismo , MAP Quinase Quinase 2/antagonistas & inibidores , MAP Quinase Quinase 2/metabolismo , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Camundongos , Morfolinas/farmacologia , Neoplasias/enzimologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Aerobic glycolysis and mitochondrial dysfunction are common features of aggressive cancer growth. We observed promoter methylation and loss of expression in neurofilament heavy polypeptide (NEFH) in a significant proportion of primary esophageal squamous cell carcinoma (ESCC) samples that were of a high tumor grade and advanced stage. RNA interference-mediated knockdown of NEFH accelerated ESCC cell growth in culture and increased tumorigenicity in vivo, whereas forced expression of NEFH significantly inhibited cell growth and colony formation. Loss of NEFH caused up-regulation of pyruvate kinase-M2 type and down-regulation of pyruvate dehydrogenase, via activation of the Akt/beta-catenin pathway, resulting in enhanced aerobic glycolysis and mitochondrial dysfunction. The acceleration of glycolysis and mitochondrial dysfunction in NEFH-knockdown cells was suppressed in the absence of beta-catenin expression, and was decreased by the treatment of 2-Deoxyglucose, a glycolytic inhibitor, or API-2, an Akt inhibitor. Loss of NEFH activates the Akt/beta-catenin pathway and increases glycolysis and mitochondrial dysfunction. Cancer cells with methylated NEFH can be targeted for destruction with specific inhibitors of deregulated downstream pathways.
Assuntos
Carcinoma de Células Escamosas/patologia , Neoplasias Esofágicas/patologia , Proteínas de Neurofilamentos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/fisiologia , beta Catenina/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Metilação de DNA , Desoxiglucose/farmacologia , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Citometria de Fluxo , Expressão Gênica , Glicólise/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Proteínas de Neurofilamentos/genética , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Transplante Heterólogo , Carga Tumoral , beta Catenina/genéticaRESUMO
Lung cancer is one of the most prevalent cancers worldwide. This study focused on small cell lung cancer (SCLC), which has a poor clinical prognosis, and attempted to elucidate potential therapeutic molecular targets. A target-specific mutational search revealed mutation of the PIK3CA gene in three of 13 SCLC cell lines and two of 15 primary SCLCs. By introducing these mutant PIK3CA cDNAs, we established artificial "PIK3CA-addicted" cells and found that Tricribine, a small-molecule inhibitor of AKT signaling that is located downstream from PIK3CA, significantly inhibited the growth and colony formation activity of these cells. Using cancer cell lines, we further showed that PIK3CA-mutated SCLC cells are more sensitive to Tricribine than PIK3CA wild-type cells. Additionally, we found that a cisplatin-resistant subclone of PIK3CA-mutant SCLC cells was equally sensitive to Tricribine. This study for the first time uncovered PIK3CA alterations in SCLC, and our findings suggest that anti-AKT molecular therapy could be effective for a subgroup of SCLC, which shows activation of specific genes, such as PIK3CA mutation, and that genetic stratification of SCLC according to the activation status of individual therapeutic target pathways could be clinically beneficial, especially for chemotherapy-resistant/relapsing tumors.
Assuntos
Antineoplásicos/farmacologia , Clorpropamida/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , Neoplasias Pulmonares/genética , Fosfatidilinositol 3-Quinases/genética , Carcinoma de Pequenas Células do Pulmão/genética , Sequência de Bases , Linhagem Celular Tumoral , Clorpropamida/farmacologia , Classe I de Fosfatidilinositol 3-Quinases , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Mutagênese Sítio-Dirigida , Mutação , Reação em Cadeia da Polimerase , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológicoRESUMO
Leptin is an adipocyte-derived hormone/cytokine that modulates immune responses. It induces functional and morphological changes in human dendritic cells (DCs), licensing them towards Th1 priming and promoting DC survival. Here we found that leptin protects DCs from spontaneous, UVB and H(2)O(2)-induced apoptosis, by triggering the activation of nuclear factor-kappa B (NF-kappaB) and a parallel up-regulation of bcl-2 and bcl-XL gene expression and Akt activation. We found that leptin activates the PI3K-Akt signaling pathway as demonstrated by the suppression of the effect of leptin on DC survival by wortmannin and API-2, which suppress the leptin-induced activation of Akt, NF-kappaB, bcl-2, bcl-XL and protection from apoptosis. These results provide insights on the immunoregulatory function of leptin, supporting a potential application in immunotherapeutic approaches.
Assuntos
Apoptose/imunologia , Células Dendríticas/imunologia , Leptina/imunologia , Fosfatidilinositol 3-Quinases/imunologia , Proteínas Proto-Oncogênicas c-akt/imunologia , Transdução de Sinais/imunologia , Androstadienos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Sobrevivência Celular/efeitos da radiação , Células Cultivadas , Clorpropamida/análogos & derivados , Clorpropamida/farmacologia , Células Dendríticas/citologia , Células Dendríticas/enzimologia , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/imunologia , Ativação Enzimática/efeitos da radiação , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Peróxido de Hidrogênio/farmacologia , Leptina/metabolismo , Leptina/farmacologia , NF-kappa B/imunologia , NF-kappa B/metabolismo , Oxidantes/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação , Células Th1/imunologia , Células Th1/metabolismo , Raios Ultravioleta , Wortmanina , Proteína bcl-X/imunologia , Proteína bcl-X/metabolismoRESUMO
Effective treatments for advanced prostate cancer are much needed. Toward this goal, we show apoptosis and impaired long-term survival of androgen-independent prostate cancer cells (PC3 and PC3 derivatives) co-treated with the cyclin-dependent kinase (CDK) inhibitor roscovitine and an AKT inhibitor (LY294002 or API-2). Apoptosis of PC3 cells by the drug combination required caspase-9 but not caspase-8 activity and thus is mitochondria-dependent. Roscovitine reduced amounts of the caspase inhibitor XIAP, and API-2 increased amounts of the BH3-only protein Bim. PC3 cells apoptosed when co-treated with API-2 and either cdk9 siRNA, dominant-negative cdk9, or the cdk9 inhibitor DRB; they did not apoptose when co-treated with API-2 and XIAP siRNA. Bax accumulated in mitochondria in response to API-2, whereas release of cytochrome c from mitochondria required both API-2 and roscovitine. We suggest that roscovitine elicits events that activate Bax once it translocates to mitochondria and that inactivation of cdk9 signals these events and the down-regulation of XIAP. Collectively, our data show apoptosis of prostate cancer cells by a drug combination and identify Bax activation as a basis of cooperation.