RESUMO
Bloodstains are frequently encountered at crime scenes and they provide important evidence about the incident, such as information about the victim or suspect and the time of death or other events. Efforts have been made to identify the age of the bloodstain's donor through genomic approaches, but there are some limitations, such as the availability of databases and the quality dependence of DNA. There is a need for the development of a tool that can obtain information at once from a small blood sample. The aim of this study is to identify bloodstain metabolite candidates that can be used to determine donor age. We prepared bloodstain samples and analyzed metabolites using high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS). Eighteen molecular features (MFs) were selected as candidates using volcano plots and multivariate analysis. Based on the MS/MS spectrum of the MFs, the following nine metabolites were identified from the METaboliteLINk database: Δ2-cis eicosenoic acid, ergothioneine, adenosine 5'-monophosphate, benzaldehyde, phenacylamine, myristic acid ethyl ester, p-coumaric acid, niacinamide, and N-arachidonoyl-L-alanine. These nine age markers at high or low abundances could be used to estimate the age of a bloodstain's donor. This study was the first to develop metabolite age markers that can be used to analyze crime scene bloodstains.
Assuntos
Manchas de Sangue , Espectrometria de Massas em Tandem , Biomarcadores , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida , HumanosRESUMO
Wound healing is initiated and progressed by complex integrated process of cellular, physiologic, and biochemical events, such as inflammation, cell migration and proliferation. Interleukin 6 (IL-6) is a multifunctional cytokine, and it could regulate the inflammatory response of wound healing process in a timely manner. Hyaluronic acid (HA) is an essential component of the extracellular matrix, and contributes significantly to cell proliferation and migration. The purpose of this study was to investigate the effects of IL-6 or/and HA on the cell migration process in human keratinocytes. Combining IL-6 and HA significantly increased the cell migration in scratch based wound healing assay. The phosphorylation of extracellular-signal-regulated kinase (ERK) was significantly increased after 1 hr of IL-6 and HA treatment, but the phosphorylation of p38 mitogen-activated protein kinase (MAPK) was not. We also found that significant increase of the NF-κB translocation from cytoplasm into nucleus after 1 hr of IL-6 or/and HA treatments. This study firstly showed that synergistic effects of combining IL-6 and HA on the cell migration of wound healing by activation of ERK and NF-κB signaling. Further studies might be required to confirm the synergistic effects of HA and IL-6 in the animal model for the development of a novel therapeutic mixture for stimulation of wound healing process.
Assuntos
Movimento Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Ácido Hialurônico/farmacologia , Interleucina-6/farmacologia , Queratinócitos/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Cicatrização , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Triglycerides (TG) are implicated in the development of atherosclerosis through formation of foam cells and induction of macrophage cell death. In this study, we report that addition of exogenous TG induced cell death in phorbol 12-myristate 13-acetate-differentiated THP-1 human macrophages. TG treatment induced a dramatic decrease in interleukin-1ß (IL-1ß) mRNA expression in a dose- and time-dependent manner. The expression of granulocyte macrophage colony-stimulating factor and platelet endothelial cell adhesion molecule remained unchanged. To identify signaling pathways involved in TG-induced downregulation of IL-1ß, we added p38 MAPK, protein kinase C (PKC) or c-Raf1 specific inhibitors. We found that inhibition of p38 MAPK alleviated the TG-induced downregulation of IL-1ß, whereas inhibition of PKC and c-Raf1 had no effect. This is the first report showing decreased IL-1ß expression during TG-induced cell death in a human macrophage line. Our results suggest that downregulation of IL-1ß expression by TG-treated macrophages may play a role during atherogenesis.
Assuntos
Morte Celular , Interleucina-1beta/metabolismo , Macrófagos/efeitos dos fármacos , Triglicerídeos/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Interleucina-1beta/genética , Macrófagos/citologia , Macrófagos/metabolismo , Camundongos , RNA Mensageiro/genética , Transdução de SinaisRESUMO
The antioxidant 3,4',5 tri-hydroxystilbene (resveratrol), a phytoalexin found in grapes, shows cancer preventive activities, including inhibition of migration and invasion of metastatic tumors. However, the molecular mechanism underlying the effect of resveratrol on tumor metastasis, especially in human metastatic lung and cervical cancers is not clear. A non-cytotoxic dosage of resveratrol causes a reduction in the generation of reactive oxygen species, and suppresses phorbol 12-myristate 13-acetate (PMA)-induced invasion and migration in both A549 and HeLa cells. Resveratrol also decreases both the expression and the enzymatic activity of matrix metalloproteinase-9 (MMP-9), and the promoter activity of PMA-stimulated MMP-9 is also inhibited. However, resveratrol does not affect either the expression or the proteolytic activity of MMP-2. Our results also show that resveratrol suppresses the transcription of MMP-9 by the inhibition of both NF-κB and AP-1 transactivation. These results indicate that resveratrol inhibits both NF-κB and AP-1 mediated MMP-9 expression, leading to suppression of migration and invasion of human metastatic lung and cervical cancer cells. Resveratrol has potential for clinical use in preventing invasion by human metastatic lung and cervical cancers.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Movimento Celular/efeitos dos fármacos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Estilbenos/farmacologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Metástase Neoplásica , Espécies Reativas de Oxigênio/metabolismo , Resveratrol , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Ativação Transcricional , Neoplasias do Colo do Útero/genéticaRESUMO
Foam cells derived from macrophages have been implicated as markers of early stage atherosclerosis development. In this study, we found that N-acetyl cysteine (NAC), a well-known inhibitor of reactive oxygen species (ROS), decreased the generation of ROS and suppressed foam cell formation in the presence of oxidized low density lipoprotein through down-regulation of cluster of differentiation 36 expression. We investigated gene expression profiles in order to determine the effects of NAC on foam cell formation using a microarray analysis. The level of apolipoprotein E, which is involved in lipid efflux, was increased and the levels of the antioxidant genes glutathione peroxidase 1 and 3 were also increased. The expression levels of the oxidative stress response and the DNA repair genes were decreased. These results were confirmed using quantitative real-time PCR. Our results indicate that oxidative stress plays an important role in foam cell formation, and that regulation of oxidation using antioxidants is a potential therapeutic method for blocking atherosclerosis development.
Assuntos
Acetilcisteína/metabolismo , Aterosclerose/metabolismo , Biomarcadores/metabolismo , Células Espumosas/citologia , Células Espumosas/metabolismo , Regulação da Expressão Gênica/fisiologia , Lipoproteínas LDL/metabolismo , Apolipoproteínas E/metabolismo , Linhagem Celular Tumoral , Primers do DNA/genética , Perfilação da Expressão Gênica , Glutationa Peroxidase/metabolismo , Humanos , Análise em Microsséries , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Glutationa Peroxidase GPX1RESUMO
The antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) is implicated in migration and invasion of metastatic tumors. However, the molecular mechanism underlying the effect of Trolox on metastatic cancer cells is not known. We found that a non-cytotoxic dose of Trolox decreased phorbol 12-myristate 13-acetate (PMA)-induced invasion and migration of both A549 and HeLa cancer cells. We also found that Trolox suppressed both the expression and the proteolytic activity of matrix metalloproteinase-9 (MMP-9), and that the promoter activity of PMA-induced MMP-9 was inhibited by Trolox. Our results show that Trolox inhibits the transcriptional activity of MMP-9 by suppression of NF-κB transactivation. These results indicate that Trolox inhibits NF-κB-mediated MMP-9 expression, leading to the suppression of migration and invasion in lung and cervical cancer cells. Trolox is a potential agent for clinical use in preventing the invasion and metastasis of human malignant lung and cervical cancers.
Assuntos
Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Movimento Celular/efeitos dos fármacos , Cromanos/farmacologia , Neoplasias Pulmonares/metabolismo , Neoplasias do Colo do Útero/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Células HeLa , Humanos , Neoplasias Pulmonares/genética , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/metabolismo , NF-kappa B/genética , Proteólise/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Ativação Transcricional/efeitos dos fármacos , Neoplasias do Colo do Útero/genéticaRESUMO
Oxygen serves as an essential factor for oxidative stress, and it has been shown to be a mutagen in bacteria. While it is well established that ambient oxygen can also cause genomic instability in cultured mammalian cells, its effect on de novo tumorigenesis at the organismal level is unclear. Herein, by decreasing ambient oxygen exposure, we report a â¼50% increase in the median tumor-free survival time of p53-/- mice. In the thymus, reducing oxygen exposure decreased the levels of oxidative DNA damage and RAG recombinase, both of which are known to promote lymphomagenesis in p53-/- mice. Oxygen is further shown to be associated with genomic instability in two additional cancer models involving the APC tumor suppressor gene and chemical carcinogenesis. Together, these observations represent the first report directly testing the effect of ambient oxygen on de novo tumorigenesis and provide important physiologic evidence demonstrating its critical role in increasing genomic instability in vivo.
Assuntos
Neoplasias Experimentais/etiologia , Oxigênio/metabolismo , Neoplasias Cutâneas/etiologia , 9,10-Dimetil-1,2-benzantraceno/toxicidade , Animais , Sequência de Bases , Western Blotting , Carcinógenos/toxicidade , Primers do DNA , Feminino , Instabilidade Genômica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Neoplasias Experimentais/induzido quimicamente , Neoplasias Cutâneas/induzido quimicamenteRESUMO
p53 regulates the cell cycle and deoxyribonucleic acid (DNA) repair pathways as part of its unequivocally important function to maintain genomic stability. Intriguingly, recent studies show that p53 can also transactivate genes involved in coordinating the two major pathways of energy generation to promote aerobic metabolism, but how this serves to maintain genomic stability is less clear. In an attempt to understand the biology, this review presents human epidemiologic data on the inverse relationship between aerobic capacity and cancer incidence that appears to be mirrored by the impact of p53 on aerobic capacity in mouse models. The review summarizes mechanisms by which p53 regulates mitochondrial respiration and proposes how this might contribute to maintaining genomic stability. Although disparate in nature, the data taken together suggest that the promotion of aerobic metabolism by p53 serves as an important tumor suppressor activity and may provide insights for cancer prevention strategies in the future.
Assuntos
Mitocôndrias/metabolismo , Neoplasias/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Aerobiose , Anaerobiose , Animais , Apoptose/fisiologia , Autofagia/fisiologia , Respiração Celular , Instabilidade Genômica , Humanos , Camundongos , Mitocôndrias/fisiologia , OxirreduçãoRESUMO
Oxygen is not only required for oxidative phosphorylation but also serves as the essential substrate for the formation of reactive oxygen species (ROS), which is implicated in ageing and tumorigenesis. Although the mitochondrion is known for its bioenergetic function, the symbiotic theory originally proposed that it provided protection against the toxicity of increasing oxygen in the primordial atmosphere. Using human cells lacking Synthesis of Cytochrome c Oxidase 2 (SCO2-/-), we have tested the oxygen toxicity hypothesis. These cells are oxidative phosphorylation defective and glycolysis dependent; they exhibit increased viability under hypoxia and feature an inverted growth response to oxygen compared with wild-type cells. SCO2-/- cells have increased intracellular oxygen and nicotinamide adenine dinucleotide (NADH) levels, which result in increased ROS and oxidative DNA damage. Using this isogenic cell line, we have revealed the genotoxicity of ambient oxygen. Our study highlights the importance of mitochondrial respiration both for bioenergetic benefits and for maintaining genomic stability in an oxygen-rich environment.
Assuntos
Respiração Celular/fisiologia , Mitocôndrias/metabolismo , Oxigênio/metabolismo , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Respiração Celular/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Citometria de Fluxo , Imunofluorescência , Células HCT116 , Humanos , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares , Fosforilação Oxidativa , Estresse Oxidativo/genética , Estresse Oxidativo/fisiologia , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismoRESUMO
OBJECTIVES: The expression of FOS, a gene critical for monocyte and macrophage function, can be inhibited by statins through the disruption of a cholesterol-independent signaling pathway. In this pilot study, we hypothesized that blood FOS mRNA levels will be sensitive to statin treatment independent of LDL cholesterol levels. METHODS: Three cohorts at increased risk of or with cardiovascular disease (CVD) were studied. Blood FOS mRNA levels were measured before and after statin treatment or in patients under stable treatment. RESULTS: Statin treatment for three months significantly reduced blood FOS mRNA and LDL cholesterol levels. However, in subjects with similar LDL levels achieved by different doses of long term statin treatment, there was an inverse relationship between statin dose and FOS expression. CONCLUSIONS: FOS mRNA levels appear to be a sensitive marker of statin treatment that is dissociated from cholesterol levels.
Assuntos
LDL-Colesterol/metabolismo , Genes fos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Proteínas Proto-Oncogênicas c-fos/sangue , Idoso , Biomarcadores/metabolismo , Proteína C-Reativa/biossíntese , Colesterol/química , Feminino , Humanos , Inflamação , Leucócitos Mononucleares/citologia , Macrófagos/citologia , Masculino , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
Cancer cells often display defects in mitochondrial respiration, thus the identification of pathways that promote cell survival under this metabolic state may have therapeutic implications. Here, we report that the targeted ablation of mitochondrial respiration markedly increases expression of Polo-like kinase 2 (PLK2) and that it is required for the in vitro growth of these nonrespiring cells. Furthermore, we identify PLK2 as a kinase that phosphorylates Ser-137 of PLK1, which is sufficient to mediate this survival signal. In vivo, knockdown of PLK2 in an isogenic human cell line with a modest defect in mitochondrial respiration eliminates xenograft formation, indicating that PLK2 activity is necessary for growth of cells with compromised respiration. Our findings delineate a mitochondrial dysfunction responsive cell cycle pathway critical for determining cancer cell outcome.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Mitocôndrias/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Animais , Apoptose , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/genética , Sobrevivência Celular/fisiologia , Transporte de Elétrons , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Immunoblotting , Camundongos , Camundongos Nus , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Chaperonas Moleculares , Neoplasias Experimentais/genética , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Consumo de Oxigênio , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas/genética , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Serina/genética , Serina/metabolismo , Transfecção , Transplante Heterólogo , Quinase 1 Polo-LikeRESUMO
RATIONALE: Exercise capacity is a physiological characteristic associated with protection from both cardiovascular and all-cause mortality. p53 regulates mitochondrial function and its deletion markedly diminishes exercise capacity, but the underlying genetic mechanism orchestrating this is unclear. Understanding the biology of how p53 improves exercise capacity may provide useful insights for improving both cardiovascular as well as general health. OBJECTIVE: The purpose of this study was to understand the genetic mechanism by which p53 regulates aerobic exercise capacity. METHODS AND RESULTS: Using a variety of physiological, metabolic, and molecular techniques, we further characterized maximum exercise capacity and the effects of training, measured various nonmitochondrial and mitochondrial determinants of exercise capacity, and examined putative regulators of mitochondrial biogenesis. As p53 did not affect baseline cardiac function or inotropic reserve, we focused on the involvement of skeletal muscle and now report a wider role for p53 in modulating skeletal muscle mitochondrial function. p53 interacts with Mitochondrial Transcription Factor A (TFAM), a nuclear-encoded gene important for mitochondrial DNA (mtDNA) transcription and maintenance, and regulates mtDNA content. The increased mtDNA in p53(+/+) compared to p53(-/-) mice was more marked in aerobic versus glycolytic skeletal muscle groups with no significant changes in cardiac tissue. These in vivo observations were further supported by in vitro studies showing overexpression of p53 in mouse myoblasts increases both TFAM and mtDNA levels whereas depletion of TFAM by shRNA decreases mtDNA content. CONCLUSIONS: Our current findings indicate that p53 promotes aerobic metabolism and exercise capacity by using different mitochondrial genes and mechanisms in a tissue-specific manner.
Assuntos
DNA Mitocondrial/metabolismo , Tolerância ao Exercício , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Mioblastos Esqueléticos/metabolismo , Esforço Físico , Proteína Supressora de Tumor p53/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Tolerância ao Exercício/genética , Glicólise/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular , Força Muscular , Mutação , Miocárdio/metabolismo , Consumo de Oxigênio , Interferência de RNA , Elementos de Resposta , Natação , Fatores de Tempo , Transdução Genética , Transfecção , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genética , Regulação para Cima , Função Ventricular EsquerdaRESUMO
Chemokines and chemokine receptors play a role in migration of circulating leukocytes to the region of inflammation. Human LZIP is an uncharacterized transcription factor and is known to participate in leukotactin (Lkn)-1/CCL15-induced cell migration. We investigated the role of human LZIP in expression of CC chemokine receptors (CCRs) and its involvement in monocyte migration. RNase protection analysis showed that LZIP increased mRNA expression of CCR2 and CCR1 in THP-1 cells. Surface expressions of both CCR2 and CCR1 were also increased by LZIP. Results from an electrophoretic mobility shift assay showed that LZIP binds to the C/EBP element in the CCR2 promoter. LZIP also enhanced the chemotactic activities of monocyte chemoattractant protein-1/CCL2 and Lkn-1. These results suggest that LZIP regulates expression of chemokine receptors that are involved in monocyte migration.
Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Movimento Celular/fisiologia , Quimiocina CCL2/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Receptores CCR2/biossíntese , Aterosclerose/tratamento farmacológico , Aterosclerose/etiologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/imunologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiocinas CC/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/imunologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/farmacologia , Humanos , Proteínas Inflamatórias de Macrófagos/farmacologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/análise , Receptores CCR1/biossíntese , Receptores CCR1/genética , Receptores CCR2/genética , Ativação Transcricional/efeitos dos fármacos , Transfecção , TransgenesRESUMO
Although Echinostoma hortense is one of the intestinal trematodes with a high infection rate in South Korea, the exact immune response against E. hortense infection has yet to be fully investigated. In the present study, we investigated differential susceptibilities in two different strains of micenamely, BALB/c (H-2d) and C3H/HeN (H-2k) mice. Likewise, we investigated the effects of ketotifen, an antiallergic drug, on the immune response against E. hortense infection. The worm recovery rate of the C3H/HeN mice was much higher than that of the BALB/c mice. The messenger ribonucleic acid (mRNA) expressions of interleukin (IL)-4 and IL-5 in the BALB/c mice were stronger than that of the C3H/HeN mice after E. hortense infection, but IL-1beta and tumor necrosis factor (TNF)-alpha expressions in the BALB/c mice were weaker than that of the C3H/HeN mice after E. hortense infection. The number of goblet cells and eosinophils increased after E. hortense infection in the BALB/c and the C3H/HeN mice. The worm recovery rate was higher and lasted longer in the ketotifen-treated mice in comparison to the untreated mice. Ketotifen suppressed the mRNA expression of IL-4 and IL-5 in the BALB/c mice, but did not in the C3H/HeN mice. The IL-1beta expressions were inhibited by ketotifen in the two strains, but TNF-alpha expression was inhibited in the C3H/HeN mice after ketotifen treatment. In addition, ketotifen inhibited the increase in eosinophils and goblet cells in varying degrees, depending on the strain. In summary, the immune sensitivity against E. hortense depends on the species of the host. The ketotifen treatment administered on the infected mice differently blocked the immune response against E. hortense infection.
Assuntos
Antialérgicos , Echinostoma/patogenicidade , Equinostomíase/tratamento farmacológico , Equinostomíase/imunologia , Animais , Antialérgicos/administração & dosagem , Antialérgicos/uso terapêutico , Citocinas/metabolismo , Echinostoma/isolamento & purificação , Equinostomíase/parasitologia , Eosinófilos/imunologia , Feminino , Células Caliciformes/imunologia , Cetotifeno/administração & dosagem , Cetotifeno/uso terapêutico , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Especificidade da Espécie , Células Th1/imunologia , Células Th2/imunologia , Resultado do TratamentoRESUMO
The genetic basis of increased glycolytic activity observed in cancer cells is likely to be the result of complex interactions of multiple regulatory pathways. Here we review the recent evidence of a simple genetic mechanism by which tumor suppressor p53 regulates mitochondrial respiration with secondary changes in glycolysis that are reminiscent of the Warburg effect. The biological significance of this regulation of the two major pathways of energy generation by p53 remains to be seen.
Assuntos
Metabolismo Energético/fisiologia , Mitocôndrias/fisiologia , Proteína Supressora de Tumor p53/fisiologia , Animais , Respiração Celular/fisiologia , Glicólise , Humanos , Neoplasias/metabolismo , Fosforilação Oxidativa , Consumo de Oxigênio/fisiologiaRESUMO
Human CC chemokine-4 (HCC-4)/CCL16 is a chemoattractant for monocytes and lymphocytes. Although HCC-4 binds to multiple CC chemokine receptors, the receptor-mediated signal transduction pathway induced by HCC-4 has not been characterized. Human osteogenic sarcoma cells stably expressing CCR1 were used to investigate HCC-4-mediated chemotaxis signaling events via CCR1. The chemotactic activity of HCC-4 as well as those of other CCR1-dependent chemokines including MIP-1alpha/CCL3, RANTES/CCL5, and Lkn-1/CCL15 was inhibited by the treatment of pertussis toxin, an inhibitor of Gi/Go protein, U73122, an inhibitor of phospholipase C (PLC), and rottlerin, a specific inhibitor of protein kinase Cdelta (PKCdelta). These results indicate that HCC-4-induced chemotaxis signaling is mediated through Gi/Go protein, PLC, and PKCdelta. SB202190, an inhibitor of p38 mitogen activated protein kinase, only blocked the chemotactic activity of HCC-4, but not those of other CCR1-dependent chemokines. SB202190 inhibited HCC-4-induced chemotaxis in a dose-dependent manner (P < 0.01). HCC-4 induces p38 activation in both a time and dose-dependent manner. However, such p38 activation was not induced by other CCR1-dependent chemokines. To further investigate the differential effect of HCC-4, the Ca2+ mobilization was examined. HCC-4 induced no intracellular Ca2+ flux in contrast to other CCR1-dependent chemokines. These results indicate that HCC-4 transduces signals differently from other CCR1-dependent chemokines and may play different roles in the immune response.
Assuntos
Quimiocinas CC/farmacologia , Quimiotaxia , Receptores de Quimiocinas/metabolismo , Acetofenonas/farmacologia , Benzopiranos/farmacologia , Cálcio/metabolismo , Estrenos/farmacologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Humanos , Toxina Pertussis/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Pirrolidinonas/farmacologia , Receptores CCR1 , Receptores de Quimiocinas/efeitos dos fármacos , Transdução de Sinais , Células Tumorais Cultivadas , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
9-cis Retinoic acid (9-CRA) is a lipophilic molecule that binds to the retinoid X receptor (RXR). Although retinoic acid (RA) has been known to regulate neutrophil differentiation, a specific role for 9-CRA in chemokine-mediated cellular processes remains obscure. We investigated the effects of 9-CRA on expression of CC chemokine receptors (CCRs) in human monocytic THP-1 cells and peripheral blood monocytes. RNase protection assay was performed to examine the mRNA levels of CCRs in 9-CRA-treated THP-1 cells. mRNA expression of CCR1 and CCR2 was induced in both a dose and time dependent manner. CCR1 and CCR2 mRNA expression began to increase from 6h after a 100nM 9-CRA treatment and reached a maximal level at 12h. Surface expression of CCRs was monitored by flow cytometry. CCR1 and CCR2 surface expression increased in 9-CRA-treated THP-1 cells, but not in untreated cells. Calcium mobilization and chemotactic activity were determined to examine the effect of 9-CRA on cell movement. The intracellular Ca(2+) concentration and the chemotactic activity increased in 9-CRA-treated cells in response to the CCR1-dependent chemokines Lkn-1, MIP-1alpha, and RANTES, and the CCR2-specific chemokine MCP-1. Increased surface expression of CCR1 and the Ca(2+) influx due to 9-CRA were confirmed in peripheral blood monocytes. Taken together, 9-CRA increases the expression levels of mRNA and protein of both CCR1 and CCR2, and the cell migration ability in THP-1 cells and peripheral blood monocytes, indicating that 9-CRA may regulate inflammatory processes through an increased response to CCR1- and CCR2-dependent chemokines.
Assuntos
Expressão Gênica/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Receptores de Quimiocinas/metabolismo , Tretinoína/farmacologia , Alitretinoína , Antineoplásicos/farmacologia , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Monócitos/metabolismo , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores CCR1 , Receptores CCR2RESUMO
Signaling molecules that bind to chemokine receptors should play key roles in regulation of cell migration induced by chemokines. To characterize the CCR1-mediated cellular signal transduction mechanism, we used the yeast two-hybrid system to identify a cellular ligand for CCR1. LZIP, which has been known as a transcription factor in various cell types, was identified as a CCR1 binding protein. Although the ability of LZIP to bind DNA is possibly what allows it to function as a transcription factor, its detailed function and participation in chemotaxis have not been established. We found that LZIP binds to CCR1 based on results of a mammalian two-hybrid assay and immunoprecipitation experiments. The 21-260 residues of LZIP were essential for interaction with CCR1. Results from a chemotaxis assay using LZIP transfected cells showed that LZIP enhanced Lkn-1-induced chemotaxis, whereas the chemotactic activities induced by other CC chemokines that bind to CCR1, including MIP-1alpha, RANTES, or HCC-4, were not affected by LZIP overexpression. These data indicate that LZIP binds to CCR1 and that the interaction between CCR1 and LZIP participates in regulation of Lkn-1-dependent cell migration without affecting the chemotactic activities of other CC chemokines that bind to CCR1.
Assuntos
Quimiocinas CC/farmacologia , Quimiotaxia/efeitos dos fármacos , Receptores de Quimiocinas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Células CHO/efeitos dos fármacos , Células CHO/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Linhagem Celular/efeitos dos fármacos , Linhagem Celular/metabolismo , Linhagem Celular Tumoral/metabolismo , Quimiocina CCL3 , Quimiocina CCL4 , Quimiocina CCL5/farmacologia , Quimiocinas CC/fisiologia , Quimiotaxia/fisiologia , Cricetinae , Cricetulus , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Humanos , Rim , Zíper de Leucina/genética , Zíper de Leucina/fisiologia , Proteínas Inflamatórias de Macrófagos/farmacologia , Dados de Sequência Molecular , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Ligação Proteica , Estrutura Terciária de Proteína , RNA Interferente Pequeno/farmacologia , Receptores CCR1 , Proteínas Recombinantes de Fusão/fisiologia , Deleção de Sequência , Transdução de Sinais/fisiologia , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética , Transfecção , Técnicas do Sistema de Duplo-HíbridoRESUMO
Recently cloned leukotactin-1 (Lkn-1) that belongs to CC chemokine family has not been characterized. To understand the intracellular events following Lkn-1 binding to CCR1, we investigated the activities of signaling molecules in response to Lkn-1 in human osteogenic sarcoma cells expressing CCR1. Lkn-1-stimulated cells showed elevated phosphorylation of extracellular signal-related kinases (ERK1/2) with a distinct time course. ERK activation was peaked in 30 min and 12 h showing biphasic activation of ERK. Pertussis toxin, an inhibitor of G(i)/G(o) protein, and phospholipase C (PLC) inhibitor blocked Lkn-1-induced activation of ERK. Protein kinase C delta (PKC delta) specific inhibitor rottlerin inhibited ERK activation in Lkn-1-stimulated cells. The activities of PLC and PKC delta were also enhanced by Lkn-1 stimulation. Dominant negative Ras inhibited activation of ERK. Immediate early response genes such as c-fos and c-myc were induced by Lkn-1 stimulation. Lkn-1 affected the cell cycle progression by cyclin D(3) induction. These results suggest that Lkn-1 activates the ERK pathway by transducing the signal through G(i)/G(o) protein, PLC, PKC delta and Ras, and it may play a role for cell proliferation, differentiation, and regulation of gene expression for other cellular processes.