RESUMO
To investigate the influence of longan flower extract (LFE) on the sensitization of colorectal cancer (CRC) cells to 5-fluorouracil (5-FU) treatment, HT-29, Colo 320DM and SW480 cells were treated with LFE and 5-FU alone and in combination, and the cell viability was then assessed by trypan blue exclusion, the cell cycle by propidium iodide staining, the mitochondria membrane potential by rhodamine 123 staining, and the expression levels of associated genes by immunoblotting and quantitative real-time polymerase chain reaction. LFE and 5-FU synergistically inhibited cell proliferation of HT-29 and Colo 320DM cells. Combined treatment also elevated the level of loss of mitochondria membrane potential of these two CRC cells and arrested HT-29 cells in the S phase of the cell cycle, in association with down-regulation of cyclin A mRNA expression. LFE synergistically potentiated chemosensitivity to 5-FU in at least two CRC cell lines. The results indicated that LFE has potential as a novel agent for the sensitization of CRC cells to 5-FU.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Sinergismo Farmacológico , Flores/química , Fluoruracila/farmacologia , Extratos Vegetais/farmacologia , Sapindaceae/química , Apoptose , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Humanos , Extratos Vegetais/químicaRESUMO
Ellagic acid has been demonstrated to inhibit the growth of several types of cancer cells. However, whether it sensitizes human colorectal carcinoma cells to 5-fluorouracil, has not yet been investigated. Colorectal carcinoma HT-29, Colo 320DM, SW480 and LoVo cells were treated with ellagic acid (2.5-25 µg/ml) and 5-fluorouracil (5-25 µM) alone and in combination and then the viability was assessed by trypan blue exclusion, apoptosis by annexin-V labeling, mitochondria membrane potential by staining with rhodamine 123, and changes in the levels of proteins involved in apoptosis by immunoblotting. Ellagic acid and 5-fluorouracil synergistically inhibited cell proliferation of HT-29, Colo 320DM and SW480 cells, but cytotoxicity toward LoVo cells seems not to be potentiated by this combination. The combination also elevated apoptotic cell death of HT-29 and Colo 320DM cells. The mitochondria membrane potential was lost in combination-treated HT-29 cells, due to increased B cell lymphoma 2-associated protein X (BAX): B cell lymphoma 2 protein (BCL-2) ratio and caspase-3 activity. Ellagic acid synergistically potentiated chemosensitivity to 5-fluorouracil in at least three colorectal cancer cell lines. The results indicate that ellagic acid has potential as a novel agent sensitizing colorectal cancer cells to 5-fluorouracil.
Assuntos
Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Ácido Elágico/farmacologia , Fluoruracila/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3/biossíntese , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteína X Associada a bcl-2/biossínteseRESUMO
Ischemia/hypoxia induces oxidative stress which is associated with neurodegenerative diseases. The present study investigated protective mechanism of carnosic acid (CA) on ischemia/reperfusion and hypoxia-induced neuronal cell injury. The results showed that CA reduced 52% of the infarct volume from brains under ischemia/reperfusion in vivo and protected the PC12 cells from hypoxic injury in vitro. CA (1.0 µM) enhanced cell viability, prevented lactic dehydrogenase (LDH) release, scavenged reactive oxygen species (ROS), increased superoxide dismutase activity, and attenuated Ca(2+) release, lipid peroxidation, and prostaglandin E2 production in hypoxic PC12 cells. In addition, CA also reduced nitric oxide (NO) and interleukine (IL)-1 and IL-6 production from activated BV-2 microglia. Furthermore, its effect on hypoxia-induced mitogen-activated protein kinases (MAPKs) signaling pathway and caspase-3 was examined. Extracellular signal-regulated protein kinases, c-jun NH2-terminal kinase, and p38 MAPK were activated during hypoxia. CA inhibited MAPKs, caspase-3, and COX-2 activation and correlated well with the diminished LDH release and apoptosis (TUNEL) in PC12 cells under hypoxia. Taken together, CA protected neuronal cells under ischemia/hypoxia through scavenging or reducing of ROS and NO, inhibiting COX-2 and MAPK pathways by anti-inflammatory and anti-oxidative properties.
Assuntos
Abietanos/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Cálcio/metabolismo , Caspase 3/genética , Caspase 3/metabolismo , Hipóxia Celular/efeitos dos fármacos , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Interleucina-1/antagonistas & inibidores , Interleucina-1/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Óxido Nítrico/antagonistas & inibidores , Óxido Nítrico/metabolismo , Células PC12 , Ratos , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
We investigated the molecular epidemiology, antibiotic susceptibility, and antimicrobial resistant gene determinants of 23 multidrug-resistant (MDR) Acinetobacter baumannii samples that were collected from 5 proximal hospitals in Taiwan during April and May 2009. Major antibiotic resistance varied from 82.6 to 100%. Fivw pulsotypes were observed to spread clonally among the 5 hospitals. PCR screening revealed high distributions of intI1 (91%), bla(OXA-23) (57%), bla(ampC) (100%), adeB (100%), adeJ (100%), and abeM (100%) genes, which were prevalent in the MDR A. baumannii isolates. Resistance gene expression was examined by reverse transcription-PCR, and showed that increased ampC expression was associated with ceftazidime resistance, but expression of adeB, adeJ, or abeM did not guarantee antimicrobial resistance phenotypes. In addition, imipenem resistance in some A. baumannii strains may be mainly modulated by genes other than bla(OXA-51-like). This is the first direct evidence indicating local spread of MDR A. baumannii in Taiwan. The resistance gene determinants are widely distributed in clonal and nonclonal-related isolates.
Assuntos
Infecções por Acinetobacter/epidemiologia , Infecções por Acinetobacter/microbiologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/genética , Infecção Hospitalar/epidemiologia , Infecção Hospitalar/microbiologia , Farmacorresistência Bacteriana Múltipla , Acinetobacter baumannii/isolamento & purificação , Antibacterianos/farmacologia , Proteínas de Bactérias/genética , Análise por Conglomerados , Eletroforese em Gel de Campo Pulsado , Perfilação da Expressão Gênica , Genótipo , Hospitais , Humanos , Testes de Sensibilidade Microbiana , Epidemiologia Molecular , Tipagem Molecular , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taiwan/epidemiologiaRESUMO
Blockage of the p53 tumor suppressor has been found to impair nerve growth factor (NGF)-induced neurite outgrowth in PC-12 cells. We report herein that such impairment could be rescued by stimulation of the A(2A) adenosine receptor (A(2A)-R), a G protein-coupled receptor implicated in neuronal plasticity. The A(2A)-R-mediated rescue occurred in the presence of protein kinase C (PKC) inhibitors or protein kinase A (PKA) inhibitors and in a PKA-deficient PC-12 variant. Thus, neither PKA nor PKC was involved. In contrast, expression of a truncated A(2A)-R mutant harboring the seventh transmembrane domain and its C terminus reduced the rescue effect of A(2A)-R. Using the cytoplasmic tail of the A(2A)-R as bait, a novel-A(2A)-R-interacting protein [translin-associated protein X (TRAX)] was identified in a yeast two-hybrid screen. The authenticity of this interaction was verified by pull-down experiments, coimmunoprecipitation, and colocalization of these two molecules in the brain. It is noteworthy that reduction of TRAX using an antisense construct suppressed the rescue effect of A(2A)-R, whereas overexpression of TRAX alone caused the same rescue effect as did A(2A)-R activation. Results of [(3)H]thymidine and bromodeoxyuridine incorporation suggested that A(2A)-R stimulation inhibited cell proliferation in a TRAX-dependent manner. Because the antimitotic activity is crucial for NGF function, the A(2A)-R might exert its rescue effect through a TRAX-mediated antiproliferative signal. This antimitotic activity of the A(2A)-R also enables a mitogenic factor (epidermal growth factor) to induce neurite outgrowth. We demonstrate that the A(2A)-R modulates the differentiation ability of trophic factors through a novel interacting protein, TRAX.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Receptor A2A de Adenosina/fisiologia , Proteína Supressora de Tumor p53/antagonistas & inibidores , Animais , Proliferação de Células/efeitos dos fármacos , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , DNA/biossíntese , Fator de Crescimento Neural/farmacologia , Células PC12 , Proteína Quinase C/fisiologia , Ratos , Receptor A2A de Adenosina/química , Transdução de SinaisRESUMO
In the present study, we used the N terminus (amino acids 1 approximately 160) of type VI adenylyl cyclase (ACVI) as bait to screen a mouse brain cDNA library and identified Snapin as a novel ACVI-interacting molecule. Snapin is a binding protein of SNAP25, a component of the SNARE complex. Co-immunoprecipitation analyses confirmed the interaction between Snapin and full-length ACVI. Mutational analysis revealed that the interaction domains of ACVI and Snapin were located within amino acids 1 approximately 86 of ACVI and 33-51 of Snapin, respectively. Co-localization of ACVI and Snapin was observed in primary hippocampal neurons. Moreover, expression of Snapin specifically eliminated protein kinase C (PKC)-mediated suppression of ACVI, but not that of cAMP-dependent protein kinase (PKA) or calcium. Mutation of the potential PKC and PKA phosphorylation sites of Snapin did not affect the ability of Snapin to reverse the PKC inhibitory effect on ACVI. Phosphorylation of Snapin by PKC or PKA therefore might not be crucial for Snapin action on ACVI. In contrast, Snapin(Delta33-51), which harbors an internal deletion of amino acids 33-51 did not affect PKC-mediated inhibition of ACVI, supporting that amino acids 33-51 of Snapin comprises the ACVI-interacting region. Consistently, Snapin exerted no effect on PKC-mediated inhibition of an ACVI mutant (ACVI-DeltaA87), which lacked the Snapin-interacting region (amino acids 1-86). Snapin thus reverses its action via direct interaction with the N terminus of ACVI. Collectively, we demonstrate herein that in addition to its association with the SNARE complex, Snapin also functions as a regulator of an important cAMP synthesis enzyme in the brain.