Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 96
Filtrar
Mais filtros








Base de dados
Intervalo de ano de publicação
1.
Mol Metab ; 85: 101947, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38677509

RESUMO

OBJECTIVE: Type 2 diabetes (T2D) is characterised by the loss of first-phase insulin secretion. We studied mice with ß-cell selective loss of the glucagon receptor (Gcgrfl/fl X Ins-1Cre), to investigate the role of intra-islet glucagon receptor (GCGR) signalling on pan-islet [Ca2+]I activity and insulin secretion. METHODS: Metabolic profiling was conducted on Gcgrß-cell-/- and littermate controls. Crossing with GCaMP6f (STOP flox) animals further allowed for ß-cell specific expression of a fluorescent calcium indicator. These islets were functionally imaged in vitro and in vivo. Wild-type mice were transplanted with islets expressing GCaMP6f in ß-cells into the anterior eye chamber and placed on a high fat diet. Part of the cohort received a glucagon analogue (GCG-analogue) for 40 days and the control group were fed to achieve weight matching. Calcium imaging was performed regularly during the development of hyperglycaemia and in response to GCG-analogue treatment. RESULTS: Gcgrß-cell-/- mice exhibited higher glucose levels following intraperitoneal glucose challenge (control 12.7 mmol/L ± 0.6 vs. Gcgrß-cell-/- 15.4 mmol/L ± 0.0 at 15 min, p = 0.002); fasting glycaemia was not different to controls. In vitro, Gcgrß-cell-/- islets showed profound loss of pan-islet [Ca2+]I waves in response to glucose which was only partially rescued in vivo. Diet induced obesity and hyperglycaemia also resulted in a loss of co-ordinated [Ca2+]I waves in transplanted islets. This was reversed with GCG-analogue treatment, independently of weight-loss (n = 8). CONCLUSION: These data provide novel evidence for the role of intra-islet GCGR signalling in sustaining synchronised [Ca2+]I waves and support a possible therapeutic role for glucagonergic agents to restore the insulin secretory capacity lost in T2D.


Assuntos
Diabetes Mellitus Tipo 2 , Glucagon , Glucose , Homeostase , Secreção de Insulina , Células Secretoras de Insulina , Receptores de Glucagon , Transdução de Sinais , Animais , Glucagon/metabolismo , Camundongos , Células Secretoras de Insulina/metabolismo , Glucose/metabolismo , Receptores de Glucagon/metabolismo , Receptores de Glucagon/genética , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Masculino , Ilhotas Pancreáticas/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dieta Hiperlipídica , Glicemia/metabolismo , Feminino
2.
Methods Cell Biol ; 133: 125-38, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27263411

RESUMO

Unraveling the structural organization of neurons can provide fundamental insights into brain function. However, visualizing neurite morphology in vivo remains difficult due to the high density and complexity of neural packing in the nervous system. Detailed analysis of neural morphology requires distinction of closely neighboring, highly intricate cellular structures such as neurites with high contrast. Green-to-red photoconvertible fluorescent proteins have become powerful tools to optically highlight molecular and cellular structures for developmental and cell biological studies. Yet, selective labeling of single cells of interest in vivo has been precluded due to inefficient photoconversion when using high intensity, pulsed, near-infrared laser sources that are commonly applied for achieving axially confined two-photon (2P) fluorescence excitation. Here we describe a novel optical mechanism, "confined primed conversion," which employs continuous dual-wave illumination to achieve confined green-to-red photoconversion of single cells in live zebrafish embryos. Confined primed conversion exhibits wide applicability and this chapter specifically elaborates on employing this imaging modality to analyze neural morphology of optically targeted single neurons in the developing zebrafish brain.


Assuntos
Microscopia Confocal/métodos , Neurônios/citologia , Análise de Célula Única/métodos , Peixe-Zebra/anatomia & histologia , Animais , Encéfalo/citologia , Embrião não Mamífero/citologia , Larva/citologia , Neuritos/ultraestrutura , Peixe-Zebra/crescimento & desenvolvimento
3.
Leukemia ; 22(10): 1899-908, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-18650843

RESUMO

Chemotherapy resistance is a major challenge in acute myeloid leukemia (AML). Besides the P-glycoprotein efflux, additional cellular factors may contribute to drug resistance in AML. c-Jun N-terminal kinase (JNK) is activated after exposure of cells to chemotherapeutics. We asked whether chemoresistance in AML is attributed to intrinsic failure of the AML blasts to activate JNK. In vitro treatment of U937 AML cell line with anthracyclines induced a rapid and robust JNK phosphorylation and apoptosis. In contrast, the anthracyline-resistant derivative cell lines U937R and URD40 showed no JNK activation after exposure to anthracyclines, also at doses that resulted in high accumulation of the drug within the cells. RNA interference-based depletion of JNK1 in drug-sensitive U937 cells made them chemoresistant, whereas selective restoration of the inactive JNK pathway in the resistant U937R cells sensitized them to anthracyclines. Short-term in vitro exposure of primary AML cells (n=29) to daunorubicin showed a strong correlation between the in vitro pharmacodymanic changes of phospho-JNK levels and the response of patients to standard induction chemotherapy (P=0.012). We conclude that JNK activation failure confers another mechanism of anthracycline resistance in AML. Elucidating the ultimate mechanisms leading to JNK suppression in chemoresistant AML may be of major therapeutic value.


Assuntos
Antraciclinas/farmacologia , Antibióticos Antineoplásicos/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/fisiologia , Leucemia Mieloide Aguda/tratamento farmacológico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Antraciclinas/uso terapêutico , Daunorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 8 Ativada por Mitógeno/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Células U937
4.
Phys Rev E Stat Nonlin Soft Matter Phys ; 75(1 Pt 1): 011901, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17358178

RESUMO

We present a general theoretical framework to discuss mechanisms of morphogen transport and gradient formation in a cell layer. Trafficking events on the cellular scale lead to transport on larger scales. We discuss in particular the case of transcytosis where morphogens undergo repeated rounds of internalization into cells and recycling. Based on a description on the cellular scale, we derive effective nonlinear transport equations in one and two dimensions which are valid on larger scales. We derive analytic expressions for the concentration dependence of the effective diffusion coefficient and the effective degradation rate. We discuss the effects of a directional bias on morphogen transport and those of the coupling of the morphogen and receptor kinetics. Furthermore, we discuss general properties of cellular transport processes such as the robustness of gradients and relate our results to recent experiments on the morphogen Decapentaplegic (Dpp) that acts in the wing disk of the fruit fly Drosophila.


Assuntos
Biofísica/métodos , Epitélio/metabolismo , Animais , Transporte Biológico , Diferenciação Celular , Membrana Celular/metabolismo , Difusão , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Cinética , Ligantes , Modelos Químicos , Modelos Estatísticos , Transdução de Sinais , Propriedades de Superfície
5.
Phytomedicine ; 14(4): 263-72, 2007 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16713222

RESUMO

In this report, we demonstrate that a 50% ethanol extract of the plant-derived product, Chios mastic gum (CMG), contains compounds which inhibit proliferation and induce death of HCT116 human colon cancer cells in vitro. CMG-treatment induces cell arrest at G(1), detachment of the cells from the substrate, activation of pro-caspases-8, -9 and -3, and causes several morphological changes typical of apoptosis in cell organelles. These events, furthermore, are time- and dose-dependent, but p53- and p21-independent. Apoptosis induction by CMG is not inhibited in HCT116 cell clones expressing high levels of the anti-apoptotic protein, Bcl-2, or dominant-negative FADD, thereby indicating that CMG induces cell death via a yet-to-be identified pathway, unrelated to the death receptor- and mitochondrion-dependent pathways. The findings presented here suggest that CMG (a) induces an anoikis form of cell death in HCT116 colon cancer cells that includes events associated with caspase-dependent pathways; and (b) might be developed into a chemotherapeutic agent for the treatment of human colon and other cancers.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fitoterapia , Pistacia , Extratos Vegetais/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/ultraestrutura , Neoplasias do Colo/tratamento farmacológico , Relação Dose-Resposta a Droga , Citometria de Fluxo , Humanos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/uso terapêutico
6.
Chemotherapy ; 52(3): 137-46, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16645270

RESUMO

OBJECTIVES: To identify parameters/factors that may contribute to the differential sensitivity to etoposide in two clones isolated from the human ovarian carcinoma SKOV-3 cell line, which does not express p53 and is resistant to platinum-based regimens. METHODS: Differential sensitivity of the cells to etoposide was monitored by microscopy to observe morphological changes, by flow cytometry analyses to detect cell cycle perturbations, and by molecular/biochemical assays to identify events involved in induction of apoptosis. RESULTS: Etoposide treatment (1) induced apoptosis in one clone, ES, but not in another clone, ER, (2) had no effect on the expression of the antiapoptotic proteins Bcl-2 and Bcl-X(L) in both cell clones, whereas the proapoptotic proteins Bak and Bax were dramatically upregulated in ES, but not ER cells, and (3) induced more extensive processing of procaspase-8, procaspase-9, and the caspase-3-targeted substrates, topoisomerase I and PARP, in ES cells. Ectopic overexpression of Bcl-2 in ES cells failed to inhibit etoposide-induced apoptosis. CONCLUSIONS: The differential susceptibility of ES and ER cells to etoposide-induced apoptosis is associated with differences in several events rather than with a specific single genetic regulator of the apoptotic machinery. We propose that the differential response of ovarian cancer patients to etoposide treatment is associated with the number of etoposide-sensitive cells in the tumor.


Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos Fitogênicos/uso terapêutico , Etoposídeo/uso terapêutico , Neoplasias Ovarianas/tratamento farmacológico , Adenocarcinoma/enzimologia , Adenocarcinoma/patologia , Antígenos de Neoplasias/metabolismo , Apoptose/efeitos dos fármacos , Caspases/metabolismo , Linhagem Celular Tumoral , Células Clonais/efeitos dos fármacos , Células Clonais/ultraestrutura , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Feminino , Citometria de Fluxo , Humanos , Neoplasias Ovarianas/enzimologia , Neoplasias Ovarianas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais
7.
Biomed Pharmacother ; 60(3): 127-33, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16527443

RESUMO

Sclareol is a labdane-type diterpene that has demonstrated a significant cytotoxic activity against human leukemic cell lines. Here, we report the effect of sclareol against the human breast cancer cell lines MN1 and MDD2 derived from the parental cell line, MCF7. MN1 cells express functional p53, whereas MDD2 cells do not express p53. Flow cytometry analysis of the cell cycle indicated that sclareol was able to inhibit DNA synthesis induce arrest at the G(0/1) phase of the cycle apoptosis independent of p53. Sclareol-induced apoptosis was further assessed by detection of fragmented DNA in the cells. Furthermore, sclareol enhanced the activity of known anticancer drugs, doxorubicin, etoposide and cisplatinum, against MDD2 breast cancer cell line.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Ciclo Celular , Proliferação de Células/efeitos dos fármacos , Diterpenos/farmacologia , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Relação Dose-Resposta a Droga , Doxorrubicina/farmacologia , Interações Medicamentosas , Etoposídeo/farmacologia , Feminino , Fase G1 , Humanos , Fase de Repouso do Ciclo Celular , Fase S , Fatores de Tempo , Proteína Supressora de Tumor p53/metabolismo
8.
Phys Rev Lett ; 94(1): 018103, 2005 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-15698137

RESUMO

We discuss the formation of graded morphogen profiles in a cell layer by nonlinear transport phenomena, important for patterning developing organisms. We focus on a process termed transcytosis, where morphogen transport results from the binding of ligands to receptors on the cell surface, incorporation into the cell, and subsequent externalization. Starting from a microscopic model, we derive effective transport equations. We show that, in contrast to morphogen transport by extracellular diffusion, transcytosis leads to robust ligand profiles which are insensitive to the rate of ligand production.


Assuntos
Diferenciação Celular/fisiologia , Fenômenos Fisiológicos Celulares , Modelos Biológicos , Morfogênese/fisiologia , Transporte Proteico/fisiologia , Fatores de Crescimento Transformadores/metabolismo , Comunicação Celular/fisiologia , Simulação por Computador , Difusão , Modelos Químicos , Fatores de Crescimento Transformadores/química
9.
Oncogene ; 20(52): 7597-609, 2001 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11753638

RESUMO

While the role of nuclear transcription factor activator protein-1 (AP-1) in cell proliferation, and of nuclear factor-kappaB (NF-kappaB) in the suppression of apoptosis are known, their role in survival of prostate cancer cells is not well understood. We investigated the role of NF-kappaB and AP-1 in the survival of human androgen-independent (DU145) and -dependent (LNCaP) prostate cancer cell lines. Our results show that the faster rate of proliferation of DU145 cells when compared to LNCaP cells correlated with the constitutive expression of activated NF-kappaB and AP-1 in DU-145 cells. The lack of constitutive expression of NF-kappaB and AP-1 in LNCaP cells also correlated with their sensitivity to the antiproliferative effects of tumor necrosis factor (TNF). TNF induced NF-kappaB activation but not AP-1 activation in LNCaP cells. In DU145 cells both c-Fos and c-Jun were expressed and treatment with TNF activated c-Jun NH2-terminal kinase (JNK), needed for AP-1 activation. In LNCaP cells, however, only low levels of c-Jun was expressed and treatment with TNF minimally activated JNK. Treatment of cells with curcumin, a chemopreventive agent, suppressed both constitutive (DU145) and inducible (LNCaP) NF-kappaB activation, and potentiated TNF-induced apoptosis. Curcumin alone induced apoptosis in both cell types, which correlated with the downregulation of the expression of Bcl-2 and Bcl-xL and the activation of procaspase-3 and procaspase-8. Overall, our results suggest that NF-kappaB and AP-1 may play a role in the survival of prostate cancer cells, and curcumin abrogates their survival mechanisms.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Curcumina/farmacologia , Regulação para Baixo , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Fator de Transcrição AP-1/metabolismo , Caspase 3 , Caspase 8 , Caspase 9 , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Precursores Enzimáticos/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Neoplasias da Próstata , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Células Tumorais Cultivadas , Fator de Necrose Tumoral alfa/farmacologia , Proteína bcl-X
10.
Cancer Res ; 61(19): 7148-54, 2001 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-11585748

RESUMO

Stimulation of CD95 leads to oligomerization of this receptor and the recruitment of the Fas-associated death domain (FADD) and procaspase-8 to form the death-inducing signaling complex (DISC). Subsequent proteolytic activation of caspase-8 at the DISC leads to the activation of downstream caspases and execution of apoptosis. The anticancer drug 9-nitrocamptothecin (9NC) inhibits the nuclear enzyme topoisomerase I (Top1), an event followed by apoptosis of cancer cells. We investigated whether other mechanisms downstream of the DNA-Top1-9NC complexing step regulate the apoptotic ability of 9NC in DU145 cells. We demonstrate that induction of apoptosis in DU145 cells, upon exposure to 9NC, is associated with de novo expression of CD95 and CD95L, suggesting that 9NC-induced apoptosis is mediated by the CD95 system. In this line, we observed early activation of procaspase-3, -7, and -8, but not -1, -9, and -10. Moreover, 9NC treatment resulted in the dramatic down-regulation of c-FLIP(short) expression, but not that of c-FLIP(long) or FADD. Furthermore, incubation of DU145 cells with a neutralizing antibody (NOK-1) to CD95L or transient transfection of a c-FLIP(short) expression vector into DU145 cells partially abrogated 9NC-triggered apoptosis. We propose that 9NC triggers apoptosis by driving DU145 cells from a nonapoptotic status (c-FLIP(short)(high), CD95(low), CD95L(low)) toward a proapoptotic status (c-FLIP(short)(low), CD95(high), CD95L(high)). These findings indicate that in addition to a Top1-mediated effect, 9NC can additionally activate a CD95/CD95L-dependent apoptotic pathway.


Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Camptotecina/farmacologia , Proteínas de Transporte/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/biossíntese , Neoplasias da Próstata/metabolismo , Receptor fas/biossíntese , Antineoplásicos/antagonistas & inibidores , Apoptose/fisiologia , Proteína Reguladora de Apoptosis Semelhante a CASP8 e FADD , Camptotecina/análogos & derivados , Camptotecina/antagonistas & inibidores , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Caspase 3 , Caspase 7 , Caspase 8 , Caspase 9 , Inibidores de Caspase , Caspases/metabolismo , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática , Precursores Enzimáticos/antagonistas & inibidores , Precursores Enzimáticos/metabolismo , Proteína Ligante Fas , Humanos , Masculino , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Transfecção , Células Tumorais Cultivadas
11.
J Biol Chem ; 276(42): 38748-54, 2001 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-11514566

RESUMO

Non-steroidal anti-inflammatory drugs (NSAIDs) are inhibitors of cyclooxygenase-1 and -2 and are useful for prevention and cure of cancers, especially colon and rectal cancers. The NSAIDs indomethacin and sulindac sulfide have been shown to induce apoptosis of colon epithelial cancer cells by a Bax-dependent mechanism that involves mitochondria-mediated activation of a caspase-9-dependent pathway. In this report, we demonstrate that indomethacin and sulindac sulfide induce apoptosis of human leukemic Jurkat cells by a mechanism that requires the Fas-associated Death Domain Protein-mediated activation of a caspase-8-dependent pathway. Therefore, NSAIDs induce apoptosis by different mechanisms depending on the cell type.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Apoptose , Proteínas de Arabidopsis , Ácidos Graxos Dessaturases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2 , Sulindaco/análogos & derivados , Western Blotting , Caspase 8 , Caspase 9 , Caspases/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Ciclo-Oxigenase 1 , Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase 2 , Inibidores de Ciclo-Oxigenase/farmacologia , Relação Dose-Resposta a Droga , Ativação Enzimática , Proteína Ligante Fas , Citometria de Fluxo , Humanos , Indometacina/farmacologia , Isoenzimas/antagonistas & inibidores , Células Jurkat , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana , Mitocôndrias/metabolismo , Modelos Biológicos , Modelos Químicos , Fenótipo , Prostaglandina-Endoperóxido Sintases , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Sulindaco/farmacologia , Fatores de Tempo , Células Tumorais Cultivadas , Proteína X Associada a bcl-2 , Receptor fas/metabolismo
12.
Anticancer Res ; 21(3B): 1823-7, 2001.
Artigo em Inglês | MEDLINE | ID: mdl-11497265

RESUMO

9-Nitrocamptothecin (9NC) results in complete regression of small-cell lung carcinoma (SCLC) and non-SCLC (NSCLC) growing as xenografts in immunodeficient mice. In this study, we have monitored histological changes in the tumors during 9NC-induced regression, and perturbations in the cell cycle of cells derived from these tumors using flow cytometry. In vivo, 9NC treatment induces dramatic changes in the tumor cells, which die by apoptosis and are ultimately eliminated from the normal tissue. In vitro, 9NC treatment resulted in apoptosis and cytostasis of the NSCLC and SCLC cells, respectively. Further, 9NC induced cytostasis in control, normal human lung fibroblasts. Therefore, the studies in vivo have indicated that 9NC acquires a remarkable antitumor activity against both the SCLC and NSCLC types tested, and that results of studies in vitro may not reflect the results observed in vivo.


Assuntos
Camptotecina/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Animais , Apoptose , Camptotecina/análogos & derivados , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma de Células Pequenas/tratamento farmacológico , Carcinoma de Células Pequenas/patologia , Ciclo Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Pulmão/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Células Tumorais Cultivadas
13.
J Med Virol ; 64(3): 238-44, 2001 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-11424110

RESUMO

The ability of the anti-cancer drug, 9-Nitrocamptothecin (9NC), to inhibit replication of HIV-1 in clinically relevant primary lymphocytic cells was studied. Primary peripheral blood lymphocytes (PBLs) from a non-infected donor were freshly infected with HIV-1 and treated with 9NC by using three different treatment schedules. Cells were monitored for cytotoxicity by the XTT metabolic cell proliferation assay and a sensitive flow cytometric assay that was capable of measuring cell cycle changes and apoptosis. 9NC inhibited replication of HIV-1 in PBLs by greater than 95% in a dose-dependent manner as measured by the level of extracellular HIV-1 p24 release. Similar results were observed, whether 9NC was applied in a single, double, or triple dose regimen. Minimal cytotoxicity was observed for both non-infected and infected PBLs, as determined by the XTT assay. Moreover, 9NC induced apoptosis within 24 hours of drug treatment in freshly infected, but not non-infected, PBLs. The data showed that 9NC reduced replication of HIV-1 in primary human lymphocytes; thus, it indicates the potential clinical utility of this drug as an alternative or adjunct therapy for HIV-infection/AIDS.


Assuntos
Fármacos Anti-HIV/farmacologia , Camptotecina/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Linfócitos/virologia , Inibidores da Transcriptase Reversa/farmacologia , Síndrome da Imunodeficiência Adquirida/tratamento farmacológico , Fármacos Anti-HIV/administração & dosagem , Apoptose/efeitos dos fármacos , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Ciclo Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Citometria de Fluxo , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Humanos , Cinética , Linfócitos/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos
14.
Cancer Res ; 61(5): 1964-9, 2001 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-11280753

RESUMO

In this study, we characterized the structure and function of topoisomerase I (top1) protein in the camptothecin (CPT)-resistant prostate cancer cell lines, DU-145/RC0.1 and DU-145/RC1 (RC0.1 and RC1, respectively). Both of the cell lines were previously selected by continuous exposure to 9-nitro-CPT. The RC0.1 and RC1 cells have high cross-resistance to CPT derivatives including SN-38 and topotecan, but are not cross-resistant to the non-top1 inhibitors etoposide, doxorubicin, and vincristine. Although the top1 protein levels were not decreased in the resistant cells compared with the parental cells, CPT-induced DNA cleavage was markedly reduced in the RC0.1 and RC1 nuclear extracts. The resistant-cell-line nuclear extracts also demonstrated top1 catalytic activity and resistance to CPT, in in vitro assays. Reverse transcription-PCR products from the resistant cell lines were sequenced, and revealed a point mutation resulting in a R364H mutation in the top1 of both RC0.1 and RC1. No wild-type top1 RNA or genomic DNA was detected in the resistant cell lines. Using a purified recombinant R364H top1, we found that the R364H mutant top1 was CPT resistant and fully active. In the published top1 crystal structure, the R364H mutation is close to the catalytic tyrosine and other well-known mutations leading to CPT resistance.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/farmacologia , DNA Topoisomerases Tipo I/genética , Inibidores Enzimáticos/farmacologia , Mutação , Neoplasias da Próstata/genética , Sequência de Aminoácidos , Sequência de Bases , Catálise , DNA Topoisomerases Tipo I/metabolismo , DNA Viral/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Masculino , Dados de Sequência Molecular , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/enzimologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Vírus 40 dos Símios/genética , Inibidores da Topoisomerase I , Células Tumorais Cultivadas
15.
Anticancer Res ; 20(5A): 2885-9, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11062697

RESUMO

In this report, we demonstrate the down-regulation of telomerase activity and c-Myc and Bcl-2 expression during 9-nitrocamptothecin (9NC)-induced regression of human DU145 prostate tumors grown as xenografts in immunodeficient mice. These changes were not observed in tumors generated by DU145-derived cells resistant to 9NC. We suggest that telomerase activity, c-Myc and Bcl-2 can collectively serve as molecular diagnostic indicators of the effectiveness of 9NC during treatment of human prostate tumors.


Assuntos
Antineoplásicos/uso terapêutico , Camptotecina/análogos & derivados , Neoplasias da Próstata/enzimologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-myc/biossíntese , Telomerase/metabolismo , Animais , Biomarcadores Tumorais , Camptotecina/uso terapêutico , Humanos , Masculino , Camundongos , Transplante de Neoplasias , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/fisiopatologia
16.
Oncogene ; 19(36): 4108-16, 2000 Aug 24.
Artigo em Inglês | MEDLINE | ID: mdl-10962571

RESUMO

We demonstrate that treatment of HCW-2 cells, an apoptotic resistant variant of the human HL-60 promyelocytic leukemia cell line with phorbol-12-myristate acetate (PMA), induced differentiation along the monocytic lineage. During this process there was a dramatic increase in the mitochondrial levels of the apoptosis effector, Bak, due to the stabilization of bak mRNA, which was correlated with the sensitization of HCW-2 cells to respond to the apoptotic effect of staurosporine (STS). Treatment of PMA-differentiated, but not undifferentiated, HCW-2 cells induced processing of Bid, substantial efflux of cytochrome c from mitochondria to the cytosol, activation of caspase-3 and apoptosis. The biological significance of the increased mitochondrial Bak in differentiated HCW-2 cells was supported by the finding that transient transfection of a bak cDNA into HCW-2 cells conferred sensitivity to STS-triggered apoptosis, as determined by pro-caspase-3 processing, cytochrome c efflux and DNA fragmentation. Our results suggest that the induction of Bak, upon monocytic differentiation, may be a critical event that regulates the apoptotic sensitivity of differentiated HCW-2 cells. Oncogene (2000) 19, 4108 - 4116


Assuntos
Apoptose , Regulação Leucêmica da Expressão Gênica , Proteínas de Membrana/genética , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3 , Northern Blotting , Western Blotting , Carcinógenos/farmacologia , Proteínas de Transporte/metabolismo , Caspase 3 , Caspases/metabolismo , Diferenciação Celular , Grupo dos Citocromos c/metabolismo , Precursores Enzimáticos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Granulócitos/citologia , Granulócitos/efeitos dos fármacos , Granulócitos/metabolismo , Células HL-60 , Humanos , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais Cultivadas , Proteína Killer-Antagonista Homóloga a bcl-2
17.
Cell Death Differ ; 7(6): 521-30, 2000 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-10822275

RESUMO

A series of bisindolylmaleimide (Bis) compounds were designed as analogs of the natural compound staurosporine (STS), which is a potent inducer of apoptosis. Many of the Bis analogs appear to be highly selective inhibitors of the protein kinase C (PKC) family, including PKC-alpha, -beta, -gamma, -delta, -epsilon, and -zeta, unlike STS, which is an inhibitor of a broad spectrum of protein kinases. In this report we describe the effects of the Bis analogs, Bis-I, Bis-II, Bis-III and Ro-31-8220 on the survival and proliferation of HL-60 cells, which have been widely used as a model cell system for studying the biological roles of PKC. Treatment of HL-60 cells with Bis-I, Bis-II, Bis-III, or Ro-31-8220 blocked phosphorylation of the PKC target protein Raf-1 with equal potency but did not appear to affect the general phosphorylation of proteins by other kinases. However, the biological effects of the Bis compounds were different: Bis-I and Bis-II had no observable effects on either cell survival or proliferation; Bis-III inhibited cell proliferation but not survival, whereas Ro-31-8220 induced apoptosis. These results indicated that the members of the PKC family which could be inhibited by the Bis analogs were required neither for survival nor proliferation of HL-60 cells. Analyses of cells treated with Ro-31-8220 showed that the apoptotic effect of Ro-31-8220 on HL-60 cells was mediated by a well-characterized transduction process of apoptotic signals: i.e., mitochondrial cytochrome c efflux and the activation of caspase-3 in the cytosol. Moreover, the ability of Ro-31-8220 to induce apoptotic activation was completely inhibited by the over-expression of the apoptotic suppressor gene, Bcl-2, in the cells. Interestingly, proliferation of the Bcl-2-over-expressing cells was still sensitive to the presence of Ro-31-8220, suggesting that the inhibitory effects of Ro-31-8220 on viability and cell proliferation were mediated by different mechanisms. In particular, the apoptotic effect of Ro-31-8220 on cells was not altered by the presence of an excess amount of the other Bis analogs, suggesting that this effect is mediated by a factor(s) other than PKC or by a mechanism which was not saturable by the other Bis analogs. Finally, structure-function analyses of compounds related to Ro-31-8220 revealed that a thioamidine prosthetic group in Ro-311-8220 was largely responsible for its apoptotic activity.


Assuntos
Apoptose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Indóis/farmacologia , Proteína Quinase C/antagonistas & inibidores , Expressão Gênica , Células HL-60 , Humanos , Indóis/química , Maleimidas/química , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Estaurosporina/análogos & derivados , Relação Estrutura-Atividade
18.
Ann N Y Acad Sci ; 922: 122-35, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11193888

RESUMO

The cytotoxicity of camptothecin (CPT) esters 1-6 was measured. Like parental camptothecin, esters 2 and 3, but not 1, 4, 5, and 6, inhibited proliferation of human leukemia cells in culture and induced programmed cell death as assessed by flow cytometry studies. Exhibition of similar levels of antiproliferative activities of CPT 2 and 3 required different incubation time periods in cell cultures, with CPT and 3 requiring the shortest and longest periods, respectively. Both 2 and 3 were inactive against cells resistant to the semisynthetic CPT derivative 9-nitrocamptothecin and unable to stabilize DNA-topoisomerase I (Topo I) "cleavable complexes" in a cell-free system, suggesting that Topo I activity was required but insufficient for the mechanism of action of 2 and 3. Mouse liver homogenate converted esters to parental CPT, but the conversion rates were different with different esters. Of four tested esters in this experiment, ester 2 had the fastest conversion rate. In vivo studies showed that ester 2 had an exceptional lack of toxicity in nude mice, even at enormous doses, and demonstrated extensive activity against human breast and colon tumors grown as xenografts in immunodeficient nude mice, whereas no antitumor activity was observed for the other esters. In conclusion, ester 2 is a prodrug of the antitumor compound CPT, and it can be administered at very high doses in mice with no appearance of toxicity. This study provides a basis for further evaluation of CPT ester 2 as an investigational anticancer agent.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camptotecina/análogos & derivados , Camptotecina/farmacologia , Inibidores Enzimáticos/farmacologia , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/toxicidade , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Camptotecina/química , Camptotecina/toxicidade , Divisão Celular/efeitos dos fármacos , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/química , Inibidores Enzimáticos/toxicidade , Ésteres/química , Ésteres/farmacologia , Ésteres/toxicidade , Feminino , Células HL-60/citologia , Células HL-60/efeitos dos fármacos , Humanos , Camundongos , Relação Estrutura-Atividade , Células U937/citologia , Células U937/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Anticancer Res ; 20(6B): 4477-82, 2000.
Artigo em Inglês | MEDLINE | ID: mdl-11205291

RESUMO

BACKGROUND: We investigated changes in the content and subcellular localization of the cell cycle regulators, cyclin B1 and cyclin-dependent kinase cdc2, in human prostate DU145 tumor and cultured cells treated with the anticancer drug 9-nitrocamptothecin (9NC). MATERIALS AND METHODS: Proteins of interest were identified by Western blot methodology using specific antibodies. RESULTS: The cyclin B1 and cdc2 contents were dramatically elevated in biopsies of DU145 tumor regressing upon 9NC-treatment. In vitro, 9NC-induced apoptosis of DU145 cells was associated with up-regulation of expression and nuclear accumulation of cyclin B1 and cdc2. No changes were observed in cyclins A and E and the cyclin-dependent kinase cdk2 in 9NC-treated DU145 tumor and cultured cells. CONCLUSION: 9NC-induced apoptosis in DU145 cells in vivo and in vitro is associated with up-regulation of expression and nuclear localization of cyclin B1 and cdc2.


Assuntos
Antineoplásicos/uso terapêutico , Proteína Quinase CDC2/metabolismo , Quinases relacionadas a CDC2 e CDC28 , Camptotecina/uso terapêutico , Ciclina B/metabolismo , Proteínas de Neoplasias/metabolismo , Apoptose , Camptotecina/análogos & derivados , Ciclina A/metabolismo , Ciclina B1 , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Quinases Ciclina-Dependentes/metabolismo , Humanos , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Indução de Remissão , Fase S/efeitos dos fármacos , Fase S/fisiologia , Células Tumorais Cultivadas/efeitos dos fármacos , Regulação para Cima
20.
Anal Biochem ; 273(1): 32-40, 1999 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-10452796

RESUMO

An analytical method is described for the quantification of S-nitrosoglutathione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmaleimide, the conversion of GSNO by 2-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricyclic isoindole derivative, and subsequent high-performance liquid chromatographic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to be identical by mass spectrometry. GSH (up to 50 microM) did not interfere with the analysis of GSNO (up to 1000 nM). The limits of detection of the method for buffered aqueous solutions of GSNO were determined as 3 nM using fluorescence and 70 nM using UV absorbance detection. Isolation of GSNO by HPLC analysis (pH 7.0) of plasma ultrafiltrate samples (200 microl) prior to derivatization allows specific and artifact-free quantification of GSNO in human and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LOQ) of the combined method was determined as 100 nM of GSNO in human plasma ultrafiltrate using fluorescence detection. No endogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 healthy humans at concentrations exceeding the LOQ of the method. After iv infusion of GSNO (125 micromol/kg body wt) in a rat for 20 min GSNO and GSH were detected in rat plasma at 60 and 130 microM, respectively. The method should be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations.


Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Glutationa/análogos & derivados , Compostos Nitrosos/sangue , o-Ftalaldeído , Acetilcisteína/metabolismo , Animais , Etilmaleimida/metabolismo , Glutationa/sangue , Glutationa/metabolismo , Humanos , Mercaptoetanol/metabolismo , Modelos Químicos , Ratos , S-Nitrosoglutationa , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA