RESUMO
Human rhinovirus (HRV) is the most common virus contributing to acute exacerbations of chronic obstructive pulmonary disease (COPD) nearly year round, but the mechanisms have not been well elucidated. Recent clinical studies suggest that high levels of growth differentiation factor 15 (GDF15) protein in the blood are associated with an increased yearly rate of all-cause COPD exacerbations. Therefore, in the current study, we investigated whether GDF15 promotes HRV infection and virus-induced lung inflammation. We first examined the role of GDF15 in regulating host defense and HRV-induced inflammation using human GDF15 transgenic mice and cultured human GDF15 transgenic mouse tracheal epithelial cells. Next, we determined the effect of GDF15 on viral replication, antiviral responses, and inflammation in human airway epithelial cells with GDF15 knockdown and HRV infection. Finally, we explored the signaling pathways involved in airway epithelial responses to HRV infection in the context of GDF15. Human GDF15 protein overexpression in mice led to exaggerated inflammatory responses to HRV, increased infectious particle release, and decreased IFN-λ2/3 (IL-28A/B) mRNA expression in the lung. Moreover, GDF15 facilitated HRV replication and inflammation via inhibiting IFN-λ1/IL-29 protein production in human airway epithelial cells. Lastly, Smad1 cooperated with interferon regulatory factor 7 (IRF7) to regulate airway epithelial responses to HRV infection partly via GDF15 signaling. Our results reveal a novel function of GDF15 in promoting lung HRV infection and virus-induced inflammation, which may be a new mechanism for the increased susceptibility and severity of respiratory viral (i.e., HRV) infection in cigarette smoke-exposed airways with GDF15 overproduction.
Assuntos
Brônquios/virologia , Células Epiteliais/virologia , Fator 15 de Diferenciação de Crescimento/metabolismo , Infecções por Picornaviridae/complicações , Pneumonia/etiologia , Rhinovirus/patogenicidade , Traqueia/virologia , Animais , Brônquios/metabolismo , Brônquios/patologia , Células Cultivadas , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Fator 15 de Diferenciação de Crescimento/genética , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Infecções por Picornaviridae/virologia , Pneumonia/metabolismo , Pneumonia/patologia , Transdução de Sinais , Traqueia/metabolismo , Traqueia/patologia , Replicação ViralRESUMO
While obesity represents one of several risk factors for colorectal cancer in humans, the mechanistic underpinnings of this association remain unresolved. Environmental stimuli, including diet, can alter the epigenetic landscape of DNA cis-regulatory elements affecting gene expression and phenotype. Here, we explored the impact of diet and obesity on gene expression and the enhancer landscape in murine colonic epithelium. Obesity led to the accumulation of histone modifications associated with active enhancers at genomic loci downstream of signaling pathways integral to the initiation and progression of colon cancer. Meanwhile, colon-specific enhancers lost the same histone mark, poising cells for loss of differentiation. These alterations reflect a transcriptional program with many features shared with the program driving colon cancer progression. The interrogation of enhancer alterations by diet in colonic epithelium provides insights into the biology underlying high-fat diet and obesity as risk factors for colon cancer.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Elementos Facilitadores Genéticos/fisiologia , Epigênese Genética/fisiologia , Regulação Neoplásica da Expressão Gênica/fisiologia , Mucosa Intestinal/fisiopatologia , Obesidade/genética , Animais , Sequência de Bases , Imunoprecipitação da Cromatina , Neoplasias Colorretais/genética , Elementos Facilitadores Genéticos/genética , Feminino , Histonas/genética , Histonas/metabolismo , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Transdução de Sinais/fisiologiaRESUMO
Non-steroidal anti-inflammatory drugs (NSAIDs) are used extensively for analgesic and antipyretic treatments. In addition, NSAIDs reduce the risk and mortality to several cancers. Their mechanisms in anti-tumorigenesis are not fully understood, but both cyclooxygenase (COX)-dependent and -independent pathways play a role. We and others have been interested in elucidating molecular targets of NSAID-induced apoptosis. In this review, we summarize updated literature regarding cellular and molecular targets modulated by NSAIDs. Among those NSAIDs, sulindac sulfide and tolfenamic acid are emphasized in this review because these two drugs have been well investigated for their anti-tumorigenic activity in many different types of cancer.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Prostaglandina-Endoperóxido Sintases/metabolismo , Animais , Humanos , Sulindaco/análogos & derivados , Sulindaco/farmacologia , ortoaminobenzoatos/farmacologiaRESUMO
OBJECTIVE: The NLRP3 inflammasome plays an important regulatory role in obesity-induced insulin resistance. NSAID activated gene-1 (NAG-1) is a divergent member of the TGF-ß superfamily. NAG-1 Tg mice are resistant to dietary- and genetic-induced obesity and have improved insulin sensitivity. The objective was to examine whether NLRP3 inflammasome activity is associated with this observed phenotype in NAG-1 Tg mice. METHODS: Key components of the NLRP3 inflammasome were examined in NAG-1 Tg mice on both regular and high fat diet (HFD) conditions. RESULTS: The expression of caspase-1 and ASC, key components of the NLRP3 inflammasome, is significantly reduced at mRNA and protein levels in white adipose tissue (WAT) of NAG-1 Tg mice. HFD increases the expression of caspase-1 and ASC in WT mice, but their expression is reduced in NAG-1 Tg mice. Furthermore, there is reduced IL-18, IL-1ß, and TNF-α expression in the WAT of NAG-1 Tg mice. NAG-1 Tg mice have significantly lower serum leptin and insulin levels and reduced expression of macrophage infiltration markers (F4/80, CD11b, and CD11c) in WAT. CONCLUSIONS: The study suggests the lower NLRP3 inflammasome activity may play a role in the resistance of NAG-1 Tg mice to diet-induced obesity and improved insulin sensitivity.
Assuntos
Proteínas de Transporte/metabolismo , Inflamassomos/metabolismo , Resistência à Insulina/genética , Obesidade/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Proteínas de Transporte/genética , Caspase 1/genética , Caspase 1/metabolismo , Dieta Hiperlipídica , Feminino , Insulina/sangue , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Leptina/sangue , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Obesidade/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Nonsteroidal anti-inflammatory drug (NSAID) activated gene-1, NAG-1, is a divergent member of the transforming growth factor-beta (TGF-ß) superfamily that plays a complex but poorly understood role in several human diseases including cancer. NAG-1 expression is substantially increased during cancer development and progression especially in gastrointestinal, prostate, pancreatic, colorectal, breast, melanoma, and glioblastoma brain tumors. Aberrant increases in the serum levels of secreted NAG-1 correlate with poor prognosis and patient survival rates in some cancers. In contrast, the expression of NAG-1 is up-regulated by several tumor suppressor pathways including p53, GSK-3ß, and EGR-1. NAG-1 expression is also induced by many drugs and dietary compounds which are documented to prevent the development and progression of cancer in mouse models. Studies with transgenic mice expressing human NAG-1 demonstrated that the expression of NAG-1 inhibits the development of intestinal tumors and prostate tumors in animal models. Laboratory and clinical evidence suggest that NAG-1, like other TGF-ß family members, may have different or pleiotropic functions in the early and late stages of carcinogenesis. Upon understanding the molecular mechanism and function of NAG-1 during carcinogenesis, NAG-1 may serve as a potential biomarker for the diagnosis and prognosis of cancer and a therapeutic target for the inhibition and treatment of cancer development and progression.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fator 15 de Diferenciação de Crescimento/metabolismo , Neoplasias/metabolismo , Animais , Epigênese Genética , Fator 15 de Diferenciação de Crescimento/genética , Humanos , Neoplasias/genéticaRESUMO
The expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) is regulated by the p53 and Egr-1 tumor suppressor pathways. Many anti-cancer drugs and chemicals induce NAG-1 expression, but the mechanisms are not fully understood. Transgenic mice expressing human NAG-1 are resistant to intestinal and prostate cancer, suggesting that NAG-1 is a tumor suppressor. Proteasome inhibitors exhibit anti-glioblastoma activities in preclinical studies. Here, we show that the proteasome inhibitors MG132 and bortezomib induced NAG-1 expression and secretion in glioblastoma cells. MG132 increased NAG-1 expression through transcriptional and post-transcriptional mechanisms. At the transcriptional level, the induction of NAG-1 required the -133 to +41 bp region of the promoter. At post-transcriptional levels, MG132 stabilized NAG-1 mRNA by increasing the half-life from 1.5 h to >8 h. Because of the dramatic increase in mRNA stability, this is likely the major contributor to MG132-mediated NAG-1 induction. Further probing into the mechanism revealed that MG132 increased phosphorylation of the p38 MAPK pathway. Consequently, inhibiting p38 phosphorylation blocked activation of the NAG-1 promoter and decreased mRNA stability, indicating that p38 MAPK activation mediates both MG132-dependent promoter activation and mRNA stabilization of NAG-1. We propose that the induction of NAG-1 by p38 MAPK is a potential contributor to the anti-glioblastoma activity of proteasome inhibitors.
Assuntos
Neoplasias Encefálicas/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Glioblastoma/metabolismo , Fator 15 de Diferenciação de Crescimento/biossíntese , Leupeptinas/farmacologia , Inibidores de Proteassoma/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Fator 15 de Diferenciação de Crescimento/genética , Humanos , Camundongos , Regiões Promotoras Genéticas/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
Nonsteroidal anti-inflammatory drug-activated gene, NAG-1, a transforming growth factor-ß member, is involved in tumor progression and development. The association between NAG-1 expression and development and progression of glioma has not been well defined. Glioblastoma cell lines have lower basal expression of NAG-1 than other gliomas and normal astrocytes. Most primary human gliomas have very low levels of NAG-1 expression. NAG-1 basal expression appeared to inversely correlate with tumor grade in glioma. Aberrant promoter hypermethylation is a common mechanism for silencing of tumor suppressor genes in cancer cells. In glioblastoma cell lines, NAG-1 expression was increased by the demethylating agent, 5-aza-2'-deoxycytidine. To investigate whether the NAG-1 gene was silenced by hypermethylation in glioblastoma, we examined DNA methylation status using genomic bisulfite sequencing. The NAG-1 promoter was densely methylated in several glioblastoma cell lines as well as in primary oligodendroglioma tumor samples, which have low basal expression of NAG-1. DNA methylation at two specific sites (-53 and +55 CpG sites) in the NAG-1 promoter was strongly associated with low NAG-1 expression. The methylation of the NAG-1 promoter at the -53 site blocks Egr-1 binding and thereby suppresses Nag-1 induction. Treatment of cells with low basal NAG-1 expression with NAG-1 inducer also did not increase NAG-1. Incubation with a demethylation chemical increased Nag-1 basal expression and subsequent incubation with a NAG-1 inducer increased NAG-1 expression. We concluded from these data that methylation of specific promoter sequences causes transcriptional silencing of the NAG-1 locus in glioma and may ultimately contribute to tumor progression.
Assuntos
Neoplasias Encefálicas/genética , Metilação de DNA , Glioblastoma/genética , Fator 15 de Diferenciação de Crescimento/genética , Anti-Inflamatórios não Esteroides/farmacologia , Apoptose/genética , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Decitabina , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Fator 15 de Diferenciação de Crescimento/biossíntese , Humanos , Ácidos Hidroxâmicos/farmacologia , Regiões Promotoras Genéticas , Sulindaco/análogos & derivados , Sulindaco/farmacologia , TransfecçãoRESUMO
BACKGROUND: Non-steroidal anti-inflammatory drug-activated gene (NAG-1), a divergent member of the transforming growth factor-beta superfamily, has been implicated in many cellular processes, including inflammation, early bone formation, apoptosis, and tumorigenesis. Recent clinical studies suggests that a C to G single nucleotide polymorphism at position 6 (histidine to aspartic acid substitution, or H6D) of the NAG-1 protein is associated with lower human prostate cancer incidence. The objective of the current study is to investigate the activity of NAG-1 H6D variant in prostate cancer tumorigenesis in vivo. METHODS: Human prostate cancer DU145 cells expressing the H6D NAG-1 or wild-type (WT) NAG-1 were injected subcutaneously into nude mice and tumor growth was monitored. Serum and tumor samples were collected for subsequent analysis. RESULTS: The H6D variant was more potent than the WT NAG-1 and inhibited tumor growth significantly compared to control mice. Mice with tumors expressing the WT NAG-1 have greater reduced both body weight and abdominal fat than mice with H6D variant tumors suggesting different activities of the WT NAG-1 and the H6D NAG-1. A significant reduction in adiponectin, leptin, and IGF-1 serum levels was observed in the tumor-bearing mice with a more profound reduction observed with expression of H6D variant. Cyclin D1 expression was suppressed in the tumors with a dramatic reduction observed in the tumor expressing the H6D variant. CONCLUSION: Our data suggest that the H6D variant of NAG-1 inhibits prostate tumorigenesis by suppressing IGF-1 and cyclin D1 expression but likely additional mechanisms are operative.
Assuntos
Fator 15 de Diferenciação de Crescimento/genética , Polimorfismo de Nucleotídeo Único , Próstata/patologia , Neoplasias da Próstata/genética , Adiponectina/sangue , Alelos , Animais , Linhagem Celular Tumoral , Ciclina D1/genética , Ciclina D1/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Leptina/sangue , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Próstata/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Transplante HeterólogoRESUMO
The antitumor effects of nonsteroidal anti-inflammatory drugs (NSAID) are assumed to be due to the inhibition of COX activity, but COX-independent mechanisms may also play an important role. NSAID-activated gene (NAG-1/GDF15) is induced by NSAIDs and has antitumorigenic activities. To determine the contribution of COX-2 inhibition and NAG-1/GDF15 expression to the prevention of colon carcinogenesis by NSAIDs, we evaluated several sulindac derivatives [des-methyl (DM)-sulindac sulfide and its prodrug DM-sulindac] that do not inhibit COX-2 activity. Sulindac sulfide and DM-sulindac induced the expression of NAG-1/GDF15 in HCT116 cells as determined by quantitative real-time PCR and Western blot. We fed APC/Min mice with 320 ppm of sulindac and doses of DM-sulindac. Only sulindac significantly inhibited tumor formation inAPC/Min mice. To determine the pharmacokinetic properties of sulindac and DM-sulindac in vivo, wild-type C57/B6 mice were fed with sulindac and DM-sulindac at 80, 160, and 320 ppm. High-performance liquid chromatography analysis revealed that the conversion of DM-sulindac to DM-sulindac sulfide (active form) was less efficient than the conversion of sulindac to sulindac sulfide (active form) in the mice. Lower levels of DM-sulindac sulfide accumulated in intestinal and colon tissues in comparison with sulindac sulfide. In addition, NAG-1/GDF15 was induced in the liver of sulindac-fed mice but not in the DM-sulindac-fed mice. Collectively, our results suggest that the tumor-inhibitory effects of sulindac in APC/Min mice may be due to, in part, NAG-1/GDF15 induction in the liver. Our study also suggests that pharmacologic properties should be carefully evaluated when developing drug candidates.
Assuntos
Anti-Inflamatórios não Esteroides/administração & dosagem , Genes APC/fisiologia , Fator 15 de Diferenciação de Crescimento/fisiologia , Pólipos Intestinais/tratamento farmacológico , Sulindaco/administração & dosagem , Animais , Anti-Inflamatórios não Esteroides/farmacocinética , Apoptose/efeitos dos fármacos , Western Blotting , Modelos Animais de Doenças , Humanos , Técnicas Imunoenzimáticas , Pólipos Intestinais/metabolismo , Pólipos Intestinais/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulindaco/farmacocinética , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Resveratrol, a dietary phytoalexin readily available in the diet, is reported to possess antitumorigenic properties in several cancers, including colorectal. However, the underlying mechanism(s) involved is not completely understood. In the present study, we investigated the effect of resveratrol treatment on gene modulation in human colorectal cancer cells and identified activating transcription factor 3 (ATF3) as the most highly induced gene after treatment. We confirmed that resveratrol upregulates ATF3 expression, both at the mRNA and protein level, and showed resveratrol involvement in ATF3 transcriptional regulation. Analysis of the ATF3 promoter revealed the importance of early growth response-1 (Egr-1; located at -245 to -236) and Krüppel-like factor 4 (KLF4; located at -178 to -174) putative binding sites in resveratrol-mediated ATF3 transactivation. Specificity of these sites to the Egr-1 and KLF4 protein was confirmed by electrophoretic mobility shift and chromatin immunoprecipitation assays. Resveratrol increased Egr-1 and KLF4 expression, which preceded ATF3 expression, and further suggests Egr-1 and KLF4 involvement in resveratrol-mediated activity. We provide evidence for Egr-1 and KLF4 interaction in the presence of resveratrol, which may facilitate ATF3 transcriptional regulation by this compound. Furthermore, we demonstrate that induction of apoptosis by resveratrol is mediated, in part, by increased ATF3 expression. Taken together, these results provide a novel mechanism by which resveratrol induces ATF3 expression and represent an additional explanation of how resveratrol exerts its antitumorigenic effects in human colorectal cancer cells.
Assuntos
Fator 3 Ativador da Transcrição/metabolismo , Anticarcinógenos/uso terapêutico , Apoptose/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Proteína 1 de Resposta de Crescimento Precoce/genética , Fatores de Transcrição Kruppel-Like/genética , Estilbenos/uso terapêutico , Sequência de Bases , Sítios de Ligação , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Luciferases/metabolismo , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Ligação Proteica , RNA Mensageiro/genética , Resveratrol , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Ativação TranscricionalRESUMO
The trans-10, cis-12 isomer of conjugated linoleic acid (CLA) causes a rapid reduction of body and adipose mass in mice. In addition to changes in adipose tissue, numerous studies have reported alterations in hepatic lipid metabolism. Livers of CLA-fed mice gain mass, partly due to lipid accumulation; however, the precise molecular mechanisms are unknown. To elucidate these mechanisms, we examined fatty acid composition and gene expression profiles of livers from a polygenic obese line of mice fed 1% trans-10, cis-12-CLA for 14 days. Analysis of gene expression data led to the identification of 1393 genes differentially expressed in the liver of CLA-fed male mice at a nominal P value of .01, and 775 were considered significant using a false discovery rate (FDR) threshold of .05. While surprisingly few genes in lipid metabolism were impacted, pathway analysis found that protein kinase A (PKA) and cyclic adenosine monophosphate (cAMP) pathways signaling pathways were affected by CLA treatment and 98 of the 775 genes were found to be regulated by hepatocyte nuclear factor 4alpha, a transcription factor important in controlling liver metabolic status.
Assuntos
Fígado Gorduroso/metabolismo , Ácidos Linoleicos Conjugados/farmacologia , Fígado/metabolismo , Obesidade/metabolismo , Animais , Fígado Gorduroso/genética , Perfilação da Expressão Gênica , Fator 4 Nuclear de Hepatócito/genética , Masculino , Camundongos , Obesidade/genéticaRESUMO
EP4 expression in human glioblastoma cells correlates with growth on soft agar. The cyclooxygenase inhibitor sulindac sulfide first altered specificity protein-1 (Sp-1) and early growth response gene-1 expression, then increased the expression of nonsteroidal anti-inflammatory drug-activated gene 1 and activating transcription factor 3, and then decreased EP4 expression. EP4 suppression was dependent on blocking the Sp-1 binding sites in the human EP4 promoter. Mutation in the Sp-1 sites in EP4 altered the promoter activity and abolished sulindac sulfide effects. The inhibitory effect of sulindac sulfide on EP4 expression was reversed by PD98059, a mitogen-activated protein/extracellular signal-regulated kinase kinase-1/extracellular signal-regulated kinase inhibitor. Sp-1 phosphorylation was dependent on sulindac sulfide-induced Erk activation. Chromatin immunoprecipitation assay confirmed that Sp-1 phosphorylation decreases Sp-1 binding to DNA and leads to the suppression of EP4. Inhibition of cell growth on soft agar assay was found to be a highly complex process and seems to require not only the inhibition of cyclooxygenase activity but also increased expression of nonsteroidal anti-inflammatory drug-activated gene 1 and activating transcription factor 3 and suppression of EP4 expression. Our data suggest that the suppression of EP4 expression by sulindac sulfide represents a new mechanism for understanding the tumor suppressor activity.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Neoplasias Encefálicas/patologia , Inibidores de Ciclo-Oxigenase/farmacologia , Glioblastoma/patologia , Receptores de Prostaglandina E/antagonistas & inibidores , Sulindaco/análogos & derivados , Fator 3 Ativador da Transcrição/metabolismo , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Imunoprecipitação da Cromatina , Ensaio de Unidades Formadoras de Colônias , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Flavonoides/farmacologia , Humanos , Imunoprecipitação , Luciferases/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de Prostaglandina E/genética , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP4 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição Sp1/metabolismo , Sulindaco/farmacologia , Células Tumorais CultivadasRESUMO
Non-steroidal anti-inflammatory drug (NSAID)-activated gene-1 (NAG-1) and COX-2 are involved in cellular processes such as inflammation, apoptosis, and tumorigenesis. To address the relationship between COX-2 and NAG-1 expression, we investigated the expression of NAG-1 and COX-2 in normal and tumor tissue from human patients, Apc(Min/+) mice, and COX-2(-/-) mice. While COX-2 expression is highly induced in tumor tissue, NAG-1 expression is reduced. Furthermore, PGE(2) reduces NAG-1 while celebrex induces NAG-1 expression. The results suggest that a possible inverse relationship exists between the expression of NAG-1 and COX-2 in tumor formation of colon tissue.
Assuntos
Neoplasias Colorretais/etiologia , Ciclo-Oxigenase 2/genética , Fator 15 de Diferenciação de Crescimento/genética , Animais , Celecoxib , Dinoprostona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Pólipos Intestinais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Pirazóis/farmacologia , Sulfonamidas/farmacologiaRESUMO
The expression of nonsteroidal anti-inflammatory drug-activated gene-1 (NAG-1) inhibits gastrointestinal tumorigenesis in NAG-1 transgenic mice (C57/BL6 background). In the present study, we investigated whether the NAG-1 protein would alter urethane-induced pulmonary lesions in NAG-1 transgenic mice on an FVB background (NAG-1(Tg+/FVB)). NAG-1(Tg+/FVB) mice had both decreased number and size of urethane-induced tumors, compared with control littermates (NAG-1(Tg+/FVB) = 16 +/- 4 per mouse versus control = 20 +/- 7 per mouse, P < 0.05). Urethane-induced pulmonary adenomas and adenocarcinomas were observed in control mice; however, only pulmonary adenomas were observed in NAG-1(Tg+/FVB) mice. Urethane-induced tumors from control littermates and NAG-1(Tg+/FVB) mice highly expressed proteins in the arachidonic acid pathway (cyclooxygenases 1/2, prostaglandin E synthase, and prostaglandin E(2) receptor) and highly activated several kinases (phospho-Raf-1 and phosphorylated extracellular signal-regulated kinase 1/2). However, only urethane-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation was decreased in NAG-1(Tg+/FVB) mice. Furthermore, significantly increased apoptosis in tumors of NAG-1(Tg+/FVB) mice compared with control mice was observed as assessed by caspase-3/7 activity. In addition, fewer inflammatory cells were observed in the lung tissue isolated from urethane-treated NAG-1(Tg+/FVB) mice compared with control mice. These results paralleled in vitro assays using human A549 pulmonary carcinoma cells. Less phosphorylated p38 MAPK was observed in cells overexpressing NAG-1 compared with control cells. Overall, our study revealed for the first time that the NAG-1 protein inhibits urethane-induced tumor formation, probably mediated by the p38 MAPK pathway, and is a possible new target for lung cancer chemoprevention.
Assuntos
Carcinógenos/toxicidade , Fator 15 de Diferenciação de Crescimento/genética , Neoplasias Pulmonares/genética , Transdução de Sinais/fisiologia , Uretana/toxicidade , Animais , Apoptose/fisiologia , Western Blotting , Linhagem Celular Tumoral , Fator 15 de Diferenciação de Crescimento/metabolismo , Humanos , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Cyclooxygenase-2 (COX-2) is up-regulated in most high-grade gliomas, and high COX-2 expression is associated with aggressive character and poor prognosis. However, the effect of COX-2 in human glioma cell lines is not well known. This study examined the effect of several stimuli, including interleukin-1beta (IL-1beta) and carcinogens, on COX-2 induction in normal astrocyte cells and human glioma cell lines U87MG, A172, and T98G. IL-1beta-induced COX-2 expression strongly at both protein and messenger ribonucleic acid levels in only the U87MG cells of the glioma cell lines. Furthermore, carcinogen induced COX-2 expression. Similar findings were also observed in normal human astrocyte cells. The U87MG glioma cell line is a good model for COX-2 induction in glioma cell lines.
Assuntos
Astrócitos/efeitos dos fármacos , Carcinógenos/farmacologia , Ciclo-Oxigenase 2/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/patologia , Interleucina-1beta/farmacologia , Proteínas de Neoplasias/biossíntese , Astrócitos/enzimologia , Benzo(a)pireno/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/enzimologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/enzimologia , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 1/genética , Ciclo-Oxigenase 2/genética , Indução Enzimática/efeitos dos fármacos , Glioma/enzimologia , Glioma/genética , Humanos , Interferon gama/farmacologia , Proteínas de Neoplasias/genética , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Acetato de Tetradecanoilforbol/farmacologia , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Nonsteroidal anti-inflammatory drug-activated gene (NAG-1) is a putative tumor suppressor whose expression can be increased by drug treatment. Glioblastoma is the most common central nervous system tumor, is associated with high morbidity and mortality, and responds poorly to surgical, chemical, and radiation therapy. The histone deacetylase inhibitors are under current consideration as therapeutic agents in treating glioblastoma. We investigated whether trichostatin A (TSA) would alter the expression of NAG-1 in glioblastoma cells. The DNA demethylating agent 5-aza-dC did not increase NAG-1 expression, but TSA up-regulated NAG-1 expression and acted synergistically with 5-aza-dC to induce NAG-1 expression. TSA indirectly increases NAG-1 promoter activity and increases NAG-1 mRNA and protein expression in the T98G human glioblastoma cell line. TSA also increases the expression of transcription factors Sp-1 and Egr-1. Small interfering RNA experiments link NAG-1 expression to apoptosis induced by TSA. Reporter gene assays, specific inhibition by small interfering RNA transfections, and chromatin immunoprecipitation assays indicate that Egr-1 and Sp-1 mediate TSA-induced NAG-1 expression. TSA also increases the stability of NAG-1 mRNA. TSA-induced NAG-1 expression involves multiple mechanisms at the transcriptional and post-transcriptional levels.
Assuntos
Inibidores Enzimáticos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma/metabolismo , Fator 15 de Diferenciação de Crescimento/biossíntese , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Proteínas de Neoplasias/biossíntese , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Linhagem Celular Tumoral , Decitabina , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fator 15 de Diferenciação de Crescimento/antagonistas & inibidores , Humanos , Proteínas de Neoplasias/antagonistas & inibidores , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , RNA Interferente Pequeno/farmacologia , Fator de Transcrição Sp1/metabolismo , Regulação para Cima/efeitos dos fármacosRESUMO
BACKGROUND & AIMS: Green tea catechins are known to have anticarcinogenic effects. Epigallocatechin-3-gallate (EGCG) accounts for almost 50% of the total catechin content in green tea extract and has very potent antioxidant effects. EGCG also inhibits angiogenesis, possibly through the inhibition of proangiogenic factors including vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which in turn, inhibits tumor growth and metastasis. However, the exact molecular mechanism by which EGCG suppresses bFGF expression is not known. Our objective was to elucidate the molecular mechanisms by which EGCG inhibits bFGF expression in colorectal cancer. METHODS: We examined posttranslational regulation of bFGF by EGCG in human colorectal cancer cells. We also examined bFGF in intestinal tumor formation of APC(Min/+) mice with and without catechin treatment. RESULTS: The bFGF protein was quickly degraded in the presence of EGCG, but a proteasome inhibitor suppressed this degradation. EGCG was also found to increase ubiquitination of bFGF and trypsin-like activity of the 20S proteasome, thereby resulting in the degradation of bFGF protein. Furthermore, EGCG suppressed tumor formation in APC(Min/+) mice, compared with vehicle-treated mice, in association with reduced bFGF expression. CONCLUSIONS: The ubiquitin-proteasome degradation pathway contributes significantly to down-regulation of bFGF expression by EGCG. Catechin compounds have fewer adverse effects than chemotherapeutic agents and hence can be used as proof-of-concept in cancer therapeutics to suppress growth and metastasis by targeting proteins such as bFGF.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Camellia sinensis , Catequina/análogos & derivados , Neoplasias Colorretais/tratamento farmacológico , Fator 2 de Crescimento de Fibroblastos/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetilcisteína/análogos & derivados , Acetilcisteína/farmacologia , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Polipose Adenomatosa do Colo/prevenção & controle , Animais , Antineoplásicos Fitogênicos/isolamento & purificação , Antineoplásicos Fitogênicos/uso terapêutico , Camellia sinensis/química , Catequina/isolamento & purificação , Catequina/farmacologia , Catequina/uso terapêutico , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/prevenção & controle , Cicloeximida/farmacologia , Inibidores de Cisteína Proteinase/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação para Baixo , Fator 2 de Crescimento de Fibroblastos/genética , Genes APC , Células HCT116 , Células HT29 , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Inibidores da Síntese de Proteínas/farmacologia , Fatores de Tempo , TransfecçãoRESUMO
NAG-1 (nonsteroidal anti-inflammatory drug-activated gene), a member of the transforming growth factor-beta superfamily, is involved in many cellular processes, such as inflammation, apoptosis/survival, and tumorigenesis. Vitamin E succinate (VES) is the succinate derivative of alpha-tocopherol and has antitumorigenic activity in a variety of cell culture and animal models. In the current study, the regulation and role of NAG-1 expression in PC-3 human prostate carcinoma cells by VES was examined. VES treatment induced growth arrest and apoptosis as well as an increase in NAG-1 protein and mRNA levels in a time- and concentration-dependent manner. VES treatment induced nuclear translocation and activation of p38 kinase. Pretreatment with p38 kinase inhibitor blocked the VES-induced increase in NAG-1 protein and mRNA levels, whereas an inhibition of protein kinase C, Akt, c-Jun NH(2)-terminal kinase, or MEK activity had no effect on VES-induced NAG-1 levels. Forced expression of constitutively active MKK6, an upstream kinase for p38, induced an increase in NAG-1 promoter activity, whereas p38 kinase inhibitor blocked MKK6-induced increase in NAG-1 promoter activity. VES treatment resulted in >3-fold increase in the half-life of NAG-1 mRNA in a p38 kinase-dependent manner and transient transfection experiment showed that VES stabilizes NAG-1 mRNA through AU-rich elements in 3'-untranslated region of NAG-1 mRNA. The inhibition of NAG-1 expression by small interfering RNA significantly blocked VES-induced poly(ADP-ribose) polymerase cleavage, suggesting that NAG-1 may play an important role in VES-induced apoptosis. These results indicate that VES-induced expression of NAG-1 mRNA/protein is regulated by transcriptional/post-transcriptional mechanism in a p38 kinase-dependent manner and NAG-1 can be chemopreventive/therapeutic target in prostate cancer.
Assuntos
Antioxidantes/farmacologia , Citocinas/genética , Neoplasias da Próstata/metabolismo , Vitamina E/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Regiões 3' não Traduzidas/genética , Antioxidantes/química , Apoptose/efeitos dos fármacos , Northern Blotting , Western Blotting , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Citocinas/antagonistas & inibidores , Citocinas/metabolismo , Imunofluorescência , Genes jun/fisiologia , Fator 15 de Diferenciação de Crescimento , Humanos , Luciferases , MAP Quinase Quinase 6/antagonistas & inibidores , MAP Quinase Quinase 6/metabolismo , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Masculino , Regiões Promotoras Genéticas/genética , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Transporte Proteico , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica/efeitos dos fármacos , Transfecção , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
For glioblastomas, COX-2 expression is linked to poor survival. COX-2 effects are mediated by the receptors EP2 and EP4, whose regulation is poorly understood. The expression of EP4, and activation or inhibition of EP4 activity in human glioblastoma T98G cells, was found to correlate with growth on soft agar. Chemoprevention drugs, troglitazone (TGZ) and some COX inhibitors, significantly suppressed EP4 expression in T98G cells in a dose dependant manner. Specificity protein 1 (Sp-1) binding sites, located within region -197 to -160 of the human EP4 promoter, are important for the transcription initiation of the human EP4 gene and are responsible for the EP4 suppression by TGZ. Mutation in the Sp-1 sites altered the promoter activity of luciferase constructs and TGZ effects on the promoter. The inhibitory effect of TGZ on EP4 expression was reversed by PD98059, a MEK-1/Erk inhibitor. Immunoprecipitation-Western blot analysis detected Sp-1 phosphorylation that was dependent on TGZ-induced Erks activation. ChIP assay confirmed that Sp-1 phosphorylation decreases its binding to DNA and as a result, leads to the suppression of EP4 expression. Thus, we propose that the expression of EP4 is regulated by Sp-1, but phosphorylation of Sp-1 induced by TGZ suppresses this expression. This represents a new and unique mechanism for the regulation of the EP4 receptor expression.
Assuntos
Antineoplásicos/farmacologia , Cromanos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores de Prostaglandina E/genética , Fator de Transcrição Sp1/metabolismo , Tiazolidinedionas/farmacologia , Astrocitoma/tratamento farmacológico , Astrocitoma/genética , Astrocitoma/metabolismo , Western Blotting , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Imunoprecipitação da Cromatina , Ensaio de Unidades Formadoras de Colônias , Inibidores de Ciclo-Oxigenase 2/farmacologia , Primers do DNA/química , Primers do DNA/genética , Flavonoides/farmacologia , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Imunoprecipitação , Luciferases/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Receptores de Prostaglandina E/antagonistas & inibidores , Receptores de Prostaglandina E/metabolismo , Receptores de Prostaglandina E Subtipo EP2 , Receptores de Prostaglandina E Subtipo EP4 , Fator de Transcrição Sp1/antagonistas & inibidores , Fator de Transcrição Sp1/genética , Transcrição Gênica , Transfecção , Troglitazona , Células Tumorais CultivadasRESUMO
Previously, we reported that non-steroidal anti-inflammatory drugs (NSAIDs) suppress cellular invasion which was mediated by thrombospondin-1 (TSP-1). As the extending study of the previous observation, we investigated the effect of NSAID-induced TSP-1 on the cellular growth and its related signaling transduction of the TSP-1 production. Among diverse NSAIDs, sulindac sulfide was most potent of inducing the human TSP-1 protein expression. Functionally, induced TSP-1 expression was associated with the growth-compensatory action of NSAID. TSP-1 expression was also elevated by mitogenic signals of ERK1/2 and RhoA GTPase pathway which had also growth-promotive capability after sulindac sulfide treatment. These findings suggest the possible mechanism through which tumor cells can survive the chemopreventive action of NSAIDs or the normal epithelium can reconstitute after NSAID-mediated ulceration in a compensatory way.