RESUMO
BACKGROUND: Bacterial contamination of platelet products is the major infectious risk in blood transfusion medicine, which can result in life-threatening sepsis in recipient. Lipocalin 2 (Lcn2) is an iron-sequestering protein in the antibacterial innate immune response, which inhibit bacterial growth. This study was aimed to evaluate the antibacterial property of Lcn2 in preventing bacterial contamination of platelets. METHODS: Recombinant Lcn2 was expressed in a eukaryotic expression system and following purification and characterization of the recombinant Lcn2, its minimum inhibitory concentration was determined. Then, platelet concentrates were inoculated with various concentrations of Staphylococcus epidermidis, Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, and Enterococcus faecalis, and the antibacterial effects of Lcn2 was evaluated at 20-24 °C. RESULTS: Results revealed that Lcn2 effectively inhibited the growth of 1.5 × 10(4) CFU/ml S. epidermidis, P. aeruginosa, K. pneumoniae, E. coli, and E. faecalis at 40 ng/ml. At this concentration, Lcn2 also inhibited the growth of 1.5 × 10(3) CFU/ml Staphylococcus aureus and Proteus mirabilis. CONCLUSION: Recombinant Lcn2 inhibited growth of a variety of platelet-contaminating bacteria. Therefore, supplementation of platelet concentrates with Lcn2 may reduce bacterial contamination.
Assuntos
Proteínas de Fase Aguda/farmacologia , Antibacterianos/farmacologia , Plaquetas/efeitos dos fármacos , Plaquetas/microbiologia , Lipocalinas/farmacologia , Transfusão de Plaquetas/métodos , Proteínas Proto-Oncogênicas/farmacologia , Enterococcus faecalis/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Humanos , Lipocalina-2 , Transfusão de Plaquetas/efeitos adversos , Pseudomonas aeruginosa/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Staphylococcus epidermidis/efeitos dos fármacosRESUMO
Drug resistance and metastasis remain major challenges in the treatment of high-risk hepatoblastoma (HB) and require the development of alternative therapeutic strategies. Modulation of apoptosis in HB cells enhances the sensitivity of these cells towards various drugs and has been discussed to enforce treatment. We investigated the impact of apoptosis sensitisers, BH3-mimetics, on the interaction between the host and HB to reduce tumour growth and dissemination while enhancing immunity. BH3-mimetics, such as obatoclax and ABT-737, enhanced the apoptosis-inducing effect of TRAIL and TNF-α resistant HB cells (HepT1 and HUH6). Tumour cell migration was inhibited by ABT-737 and more markedly by obatoclax. In an orthotopic model of HB, tumour uptake was reduced when the cells were pretreated with low concentrations of obatoclax. Only 1 of 7 mice developed HB in the liver, compared with an incidence of 0.8 in the control group. In summary, our study showed that apoptosis sensitisers had broader effects on HB cells than expected including migration and susceptibility to cytokines in addition to the known effects on drug sensitization. Sensitising HB to apoptosis may also allow resistant HB to be targeted by immune cells and prevent tumour cell dissemination.
Assuntos
Materiais Biomiméticos/farmacologia , Compostos de Bifenilo/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Hepatoblastoma/prevenção & controle , Neoplasias Hepáticas/prevenção & controle , Nitrofenóis/farmacologia , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , Pirróis/farmacologia , Sulfonamidas/farmacologia , Animais , Materiais Biomiméticos/química , Compostos de Bifenilo/química , Transformação Celular Neoplásica/patologia , Células Cultivadas , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Hepatoblastoma/patologia , Humanos , Indóis , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos Nus , Camundongos Transgênicos , Nitrofenóis/química , Fragmentos de Peptídeos/química , Piperazinas/química , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas/química , Pirróis/química , Sulfonamidas/químicaRESUMO
Although tamoxifen treatment is associated with improved survival in patients with estrogen receptor (ER)-positive breast tumors, resistance remains an important clinical obstacle. Signaling through growth factor signaling pathways, in particular through receptor tyrosine kinases, has been demonstrated to confer tamoxifen resistance in an estradiol-independent manner. The Ron receptor tyrosine kinase, a member of the c-Met family of receptors, is expressed in a number of human epithelial tumors, and elevated expression of Ron is associated with poor prognosis in women with breast cancer. In this report, we evaluated the role of Ron receptor activation in conferring resistance to tamoxifen in human and murine breast cancer cell lines. Activation of Ron by its ligand, hepatocyte growth factor-like protein (HGFL) was associated with partial rescue from tamoxifen-induced growth inhibition in Ron-expressing cell lines. Western analysis revealed that treatment of the T47D human breast cancer cell line with tamoxifen and HGFL was associated with increased phosphorylation of mitogen-activated protein kinase (MAPK) 1/2 and phosphorylation of serine residue 118 of ER. Expression of ER-dependent genes was increased in cells treated with tamoxifen and HGFL by quantitative reverse transcription-polymerase chain reaction. All of these effects were inhibited by treatment with either a Ron-neutralizing antibody or a MEK1 inhibitor, suggesting the specificity of the effect to Ron, and the involvement of the MAPK 1/2 signaling pathway. In summary, these results illustrate a novel connection between the Ron receptor tyrosine kinase and an important mechanism of tamoxifen resistance in breast cancer.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Carcinoma/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores Proteína Tirosina Quinases/agonistas , Tamoxifeno/uso terapêutico , Animais , Antineoplásicos Hormonais/uso terapêutico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos/genética , Ativação Enzimática/efeitos dos fármacos , Feminino , Flavonoides/farmacologia , Fator de Crescimento de Hepatócito/farmacologia , Humanos , Camundongos , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas/farmacologia , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Estrogênio/genética , Receptores de Estrogênio/metabolismo , TransfecçãoRESUMO
Recent studies establish a critical role of selenium in cancer prevention in vitro and in vivo. Selenium may sensitize TRAIL-mediated apoptosis in human renal cancer cells and increase therapeutic efficacy. In this study, we demonstrate that concomitant administration of TRAIL and Se-methylselenocysteine (Se-MSC) produces synergistic effects on the induction of apoptosis in Caki cells. Se-MSC rapidly and specifically down-regulates expression of the Bcl-2 at transcriptional level. The forced expression of Bcl-2 attenuated Se-MSC plus TRAIL-mediated apoptosis, suggesting that the lessened Bcl-2 expression caused by Se-MSC treatment is critical to the increased sensitivity to TRAIL in renal cancer cells. In addition, we demonstrate that the synergistic effects of Se-MSC and TRAIL result from the activation of the caspase-dependent pathways. Co-administration of HA14-1, a small molecule Bcl-2 inhibitor and TRAIL increased apoptosis in Caki cells. Taken together, Se-MSC-mediated down-regulation of Bcl-2 is able to sensitize Caki cells for TRAIL-induced apoptosis. Thus, selenium-based dietary compounds may help to overcome resistance to TRAIL-mediated apoptosis in renal cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Cisteína/análogos & derivados , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes bcl-2 , Compostos Organosselênicos/farmacologia , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Caspases/metabolismo , Caspases/fisiologia , Cisteína/administração & dosagem , Cisteína/farmacologia , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Sinergismo Farmacológico , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Compostos Organosselênicos/administração & dosagem , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacologia , Proteínas Proto-Oncogênicas/administração & dosagem , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Selenocisteína/análogos & derivados , Ligante Indutor de Apoptose Relacionado a TNF/administração & dosagem , Células Tumorais CultivadasRESUMO
In order to investigate developmental processes, several methods have been established that allow the visualization of local proliferation zones and to follow their dynamics during morphogenesis. In this study we present a detailed description of transitory and continuous proliferation zones in the developing chick embryo. By tracing the S-phase marker proliferating cell nuclear antigen (PCNA) at the mRNA level we were able to identify the initiation and termination of proliferation programs. This approach provides additional information in comparison to the well-known BrdU incorporation or the PCNA immunostaining, which exclusively labels cells that contain PCNA protein. By means of PCNA in situ hybridization we analyzed the normal expression pattern in the 2- to 5-day-old chick embryo. We furthermore monitored the effects on PCNA expression after various manipulations such as removal of the apical ectodermal ridge (AER), the zone of polarizing activity (ZPA), and the surface ectoderm. In addition, we applied morphogens, such as fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs), and retinoic acid (RA), and subsequently analyzed changes in the pattern of PCNA expression. While ablation of ZPA, AER, or ectoderm are known to reduce cell proliferation and were paralleled by loss of PCNA expression, neither BMP-2 nor BMP-4 affected PCNA expression. Upregulation of PCNA expression could be achieved by application of RA or FGFs, factors known to induce cell proliferation during limb bud outgrowth. The PCNA in situ hybridization data presented here clearly show that this method offers a novel, very sensitive tool for tracing cell proliferation and for visualizing the dynamic patterns arising due to the initiation and termination of the proliferation program.
Assuntos
Proliferação de Células , Antígeno Nuclear de Célula em Proliferação/genética , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 2 , Proteína Morfogenética Óssea 4 , Proteínas Morfogenéticas Ósseas/farmacologia , Bromodesoxiuridina/análise , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , DNA Complementar/isolamento & purificação , Fator 4 de Crescimento de Fibroblastos , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Imuno-Histoquímica , Hibridização In Situ , Botões de Extremidades/embriologia , Dados de Sequência Molecular , Antígeno Nuclear de Célula em Proliferação/análise , Proteínas Proto-Oncogênicas/farmacologia , RNA Mensageiro/análise , Alinhamento de Sequência , Fator de Crescimento Transformador beta/farmacologia , TretinoínaRESUMO
We have isolated a chick Twist gene (cTwist) and examined its expression pattern during development by whole mount in situ hybridization. In early embryos, cTwist transcripts are found in the developing somites, lateral plate mesoderm, limb mesenchyme, branchial arches and head mesenchyme. At later stages, cTwist expression is found in the sclerotome and dermatome, limb bud mesenchyme, interdigital regions, and distal mesenchyme of the maxillary and mandibular processes. In the developing feathers, cTwist is expressed in the mesenchyme of the buds and becomes restricted to the proximal region of the feather filaments. Additionally, we report that the expression of cTwistin the limb mesenchyme is regulated by the AER, FGFs, RA and SHH. The FGFs secreted by the AER seem to have a critical role in maintaining cTwist expression. SHH is also able to maintain cTwist expression but only in the presence of the AER. Overall, our results provide new evidence that reinforce the existence of an interplay between the cTwist and FGF signalling pathways.
Assuntos
Extremidades/embriologia , Fatores de Transcrição/genética , Sequência de Aminoácidos , Animais , Proteínas Aviárias , Sequência de Bases , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Embrião de Galinha , Clonagem Molecular , DNA Complementar/genética , Fator 4 de Crescimento de Fibroblastos , Fator 8 de Crescimento de Fibroblasto , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Proteínas Hedgehog , Hibridização In Situ , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Homologia de Sequência de Aminoácidos , Transativadores/fisiologia , Tretinoína/farmacologia , Proteína 1 Relacionada a TwistRESUMO
Many non-Hodgkins B-cell lymphomas possess a deregulated bcl-2 gene resulting in a phenotype that is apparently resistant to programmed cell death (apoptosis). We have used a mouse lymphoma cell line (S49.1) that undergoes apoptosis in response to a variety of stimuli to determine the effect of bcl-2 expression on induction of apoptosis. S49 cells were stably transfected with recombinant amphotrophic retroviruses carrying either a G418 antibiotic resistance gene alone (S49-NEO) or this gene in combination with a bcl-2 complementary DNA (S49-Bcl-2). Three different agents previously shown to activate apoptosis by different pathways in S49 cells (dexamethasone, the calcium ionophore A23187, and cycloheximide) were used to examine the effect of bcl-2 expression on cell growth and apoptosis caused by multiple signal transduction pathways. Dexamethasone (DEX) treatment inhibited cell growth and stimulated cell death in S49-NEO cells. Although S49-Bcl-2 cells exhibited a similar antiproliferative response, they failed to die in response to steroid treatment. Western blot analysis revealed no difference in the levels of glucocorticoid receptor protein in the two cell lines, and both responded to glucocorticoid with a profound inhibition of protein synthesis. Cycloheximide (CX) and A23187 also had antiproliferative and cell killing effects in both cell types, although higher concentrations of each agent were needed to kill S49-Bcl-2 cells. To determine whether the loss of viability in response to these drugs was due to apoptosis, cells were examined morphologically and DNA integrity was examined by gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Apoptose/efeitos dos fármacos , Calcimicina/farmacologia , Cicloeximida/farmacologia , Glucocorticoides/farmacologia , Linfoma/patologia , Proteínas Proto-Oncogênicas/farmacologia , Animais , Morte Celular/efeitos dos fármacos , DNA/metabolismo , Dexametasona/farmacologia , Resistência a Medicamentos/genética , Camundongos , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2 , Receptores de Glucocorticoides/fisiologia , Transdução de Sinais , Transfecção , Células Tumorais CultivadasRESUMO
It has previously been reported that PDGF isoforms AB and BB induce an increase of cytosolic free calcium in cultured bovine lens epithelial cells in a dose-dependent manner. To evaluate the biological capacity of PDGF, we investigated the proliferative response of bovine lens epithelial cells to stimulation with PDGF-AA, -AB and -BB. Since various unspecific components of serum-containing media act as mitogenes and mask the effect of PDGF, serum-free culture conditions were a prerequisite for growth-factor-induced effects. Therefore, a basic medium (Waymouth's MB 752/1 with Ham F12 Nutrient Mixture 1:2, v/v; Gibco BRL) was supplemented with only 2 mM CaCl2, 10 micrograms/ml Transferrin, 10 micrograms/ml Thyroglobulin (both Sigma Chemie) and standard amounts of antibiotics. PDGF isoforms were obtained by separate expression of cloned genes in Escherichia coli, which has been previously described. Under these conditions the isoforms PDGF-AB and -BB caused an increase in proliferation in a dose-dependent manner. The maximum increase of the cell number was 21% for PDGF-AB with an EC5 of 5 ng/ml. PDGF-BB revealed a maximum increase of 33% with an EC5 of 1.5 ng/ml. PDGF-AA, when used in similar concentration was ineffective. These data show the involvement of PDGF isoforms AB and BB in the replicative action of BLEC.