RESUMO
Growing evidence suggests that genetic and epigenetic factors, including environmental factors, contribute to the development of oral squamous cell carcinoma (OSCC). Here, we investigated the transcriptional silencing of the CD24, CD44, CD133, and CD147 genes, which are well-known cancer stem cell surface markers in various cancer types, including OSCC. We first examined the correlation between the transcriptional expression level and reactivation by 5-aza-2'-deoxycytidine (5-aza-dC) and the promoter methylation levels of the four genes in several OSCC cell lines. We observed promoter hypermethylation for the CD24, CD133, and CD147 genes at 70%, 75%, and 70%, respectively, in OSCC cell lines compared to normal oral mucosa tissues (<53%), indicating that this methylation pattern is cancer-specific, which was confirmed by bisulfite sequencing analysis. More specifically, the expression and methylation profiles of CD133 and CD147 extracted from The Cancer Genome Atlas (TCGA) database were negatively correlated, supporting their epigenetic regulation in primary OSCC tumors. The methylation status of CD133 and CD147 was associated with poor survival in patients with OSCC using the TCGA database. Our findings provide additional insight into the abnormal DNA methylation of CD133 and that CD147 could be used for the diagnosis and therapeutic treatment of patients with OSCC.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Humanos , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Carcinoma de Células Escamosas/diagnóstico , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Metilação de DNA , Células-Tronco Neoplásicas/patologia , Neoplasias de Cabeça e Pescoço/genéticaRESUMO
Myeloid cell leukemia sequence 1 (MCL1), an antiapoptotic Bcell lymphoma 2 (BCL2) family molecule frequently amplified in various human cancer cells, is known to be critical for cancer cell survival. MCL1 has been recognized as a target molecule for cancer treatment. While various agents have emerged as potential MCL1 blockers, the present study presented acriflavine (ACF) as a novel MCL1 inhibitor in triplenegative breast cancer (TNBC). Further evaluation of its treatment potential on lung adenocarcinoma and glioblastoma multiforme (GBM) was also investigated. The anticancer effect of ACF on TNBC cells was demonstrated when MDAMB231 and HS578T cells were treated with ACF. ACF significantly induced typical intrinsic apoptosis in TNBCs in a dose and timedependent manner via MCL1 downregulation. MCL1 downregulation by ACF treatment was revealed at each phase of protein expression. Initially, transcriptional regulation via reverse transcriptionquantitative PCR was validated. Then, posttranslational regulation was explained by utilizing an inhibitor against protein biosynthesis and proteasome. Lastly, immunoprecipitation of ubiquitinated MCL1 confirmed the posttranslational downregulation of MCL1. In addition, the synergistic treatment efficacy of ACF with the wellknown MCL1 inhibitor ABT263 against the TNBC cells was explored [combination index (CI)<1]. Conjointly, the anticancer effect of ACF was assessed in GBM (U87, U251 and U343), and lung cancer (A549 and NCIH69) cell lines as well, using immunoblotting, cytotoxicity assay and FACS. The effect of the combination treatment using ACF and ABT263 was estimated in GBM (U87, U343 and U251), and nonsmall cell lung cancer (A549) cells likewise. The present study suggested a novel MCL1 inhibitory function of ACF and the synergistic antitumor effect with ABT263.
Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Glioblastoma/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Acriflavina/farmacologia , Acriflavina/uso terapêutico , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Linhagem Celular Tumoral/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Combinação de Medicamentos , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/uso terapêutico , Sulfonamidas/farmacologia , Sulfonamidas/uso terapêuticoRESUMO
Inflammatory bowel disease is known to be associated with a genetic predisposition involving multiple genes; however, there is growing evidence that abnormal interactions with environmental factors, particularly epigenetic factors, can also significantly contribute to the development of inflammatory bowel disease (IBD). Although many genome-wide association studies have been performed to identify the genetic changes underlying the pathogenesis of Crohn's disease, the role of epigenetic alterations based on molecular complications arising from Crohn's disease (CD) is poorly understood. We employed an unbiased approach to define DNA methylation alterations in colonoscopy samples from patients with CD using the HumanMethylation450K BeadChip platform. Technical and functional validation was performed by methylation-specific PCR (MSP) and bisulfite sequencing of a validation set of 207 patients with CD samples. Immunohistochemistry (IHC) analysis was performed in the representative sample sets. DNA methylation profile in CD revealed that 135 probes (24 hypermethylated and 111 hypomethylated probes) were differentially methylated. We validated the methylation levels of 19 genes that showed hypermethylation in patients with CD compared with normal controls. We uniquely identified that the fragile histidine triad (FHIT) gene was hypermethylated in a disease-specific manner and its protein level was downregulated in patients with CD. Pathway analysis of the hypermethylated candidates further suggested putative molecular interactions relevant to IBD pathology. Our data provide information on the biological and clinical implications of DNA hypermethylated genes in CD, identifying FHIT methylation as a promising new biomarker for CD. Further study of the role of FHIT in IBD pathogenesis may lead to the development of new therapeutic targets.
RESUMO
Background: Arsenic compounds (As2O3 and As4O6) have demonstrated anticancer effects in various malignancies. In this study, the cytotoxicity of arsenic compounds on ovarian cancer cell lines and the anticancer activity of the combination of arsenic compounds and cisplatin IN chemoresistant ovarian cancer cells were investigated.Methods: We investigated the cytotoxicity of As2O3 and As4O6 and their combinations with cisplatin in the paclitaxel-sensitive ovarian cancer cell lines SKOV3ip1 and HeyA8 and paclitaxel-resistant ovarian cancer cell lines SKOV3TRip2 and HeyA8-MDR. Growth and apoptosis were evaluated by MTT assay and annexin V assay using flow cytometry, respectively. For detection of apoptotic cells, immunofluorescence was performed using a cleaved caspase-3 antibody. Cell-cycle distribution was determined by propidium iodide staining and flow cytometry.Results: Treatment of each cell line with As2O3 or As4O6 led to a marked dose-dependent inhibition of cell growth. As2O3 and As4O6 treatment induced caspase-3-dependent apoptosis in all cell lines compared to the respective control groups (p < .05). As2O3 and As4O6 induced apoptosis of paclitaxel-sensitive and -resistant cancer cell lines following G2/M cell cycle arrest (p < .05). A synergistic effect was achieved by combining cisplatin with As2O3 or As4O6 in the paclitaxel-resistant ovarian cancer cell lines.Conclusions: As2O3 and As4O6 can inhibit cell growth and induce apoptosis in paclitaxel-sensitive and -resistant ovarian cancer cell lines. Their combination with cisplatin resulted in a synergistic effect in paclitaxel-resistant cancer cell lines. These results suggest that arsenic compounds may be given in monotherapy or combination therapy with cisplatin for treating paclitaxel-resistant ovarian cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Trióxido de Arsênio/farmacologia , Cisplatino/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Trióxido de Arsênio/uso terapêutico , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cisplatino/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Feminino , Citometria de Fluxo , Humanos , Paclitaxel/farmacologiaRESUMO
The lung is a prototypic organ that was evolved to reduce immunopathology during the immune response to potentially hazardous endogenous and exogenous antigens. In this study, we show that donor CD4+ T cells transiently induced expression of indoleamine 2,3-dioxygenase (IDO) in lung parenchyma in an IFN-γ-dependent manner early after allogeneic hematopoietic stem cell transplantation (HSCT). Abrogation of host IDO expression by deletion of the IDO gene or the IFN-γ gene in donor T cells or by FK506 treatment resulted in acute lethal pulmonary inflammation known as idiopathic pneumonia syndrome (IPS). Interestingly, IL-6 strongly induced IDO expression in an IFN-γ-independent manner when deacetylation of STAT3 was inhibited. Accordingly, a histone deacetylase inhibitor (HDACi) could reduce IPS in the state where IFN-γ expression was suppressed by FK506. Finally, l-kynurenine produced by lung epithelial cells and alveolar macrophages during IPS progression suppresses the inflammatory activities of lung epithelial cells and CD4+ T cells through the aryl hydrocarbon receptor pathway. Taken together, our results reveal that IDO is a critical regulator of acute pulmonary inflammation and that regulation of IDO expression by HDACi may be a therapeutic approach for IPS after HSCT.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Transplante de Células-Tronco Hematopoéticas , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Pneumonia/metabolismo , Receptores de Hidrocarboneto Arílico/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Feminino , Doença Enxerto-Hospedeiro , Transplante de Células-Tronco Hematopoéticas/mortalidade , Inibidores de Histona Desacetilases/farmacologia , Indolamina-Pirrol 2,3,-Dioxigenase/genética , Interferon gama/genética , Interferon gama/metabolismo , Interferon gama/farmacologia , Cinurenina/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Pneumonia/tratamento farmacológico , Receptores de Hidrocarboneto Arílico/imunologia , Receptores de Interferon/genética , Receptores de Interferon/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Linfócitos T Reguladores/imunologia , Tacrolimo/farmacologia , Receptor de Interferon gamaRESUMO
Pulmonary fibrosis, a potentially fatal disease, results from acute and chronic interstitial lung diseases. Fucoxanthin (Fx), a carotenoid found in brown seaweed, shows a wide range of pharmacological activities. In this study, we investigated the antifibrotic effects of fucoxanthin and their underlying molecular mechanisms in transforming growth factor-beta1 (TGF-ß1)-stimulated human pulmonary fibroblasts (HPFs). Thus, the effects of Fx on TGF-ß1-induced expression of fibrotic factors, such as alpha-smooth muscle actin (α-SMA), type 1 collagen, fibronectin, and interleukin-6 (IL-6), in HPFs were investigated. We performed an enzyme-linked immunosorbent assay (ELISA), and a western blot analysis to elucidate the mechanisms underlying the antifibrotic effects of Fx in TGF-ß1-stimulated cells. The contractile activity of HPFs was measured using a collagen gel contraction assay. We also investigated the effects of Fx on inflammation and fibrosis in bleomycin (BLM)-induced pulmonary fibrosis mouse model. We observed that Fx inhibited the TGF-ß1-induced expression of α-SMA, type 1 collagen, fibronectin, and IL-6 in HPFs. Similarly, markedly inhibition of TGF-ß1-induced phosphorylation of p-38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3K)/Akt, and Smad2/Smad3 (Smad2/3) was observed after Fx treatment. Collagen contraction also significantly decreased on fucoxanthin treatment. Intraperitoneal injection of Fx (10mg/kg) in mice inhibited BLM-induced lung fibrosis and type I collagen protein expression. Overall, our findings suggest that Fx may be effective in the treatment of pulmonary fibrosis owing to its potent antifibrotic activity.
Assuntos
Bleomicina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/metabolismo , Xantofilas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Humanos , Interleucina-6/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , Ratos , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Xantofilas/uso terapêuticoRESUMO
Cystatin SN (CST1), a known inhibitor of cathepsin B (CatB), has important roles in tumor development. Paradoxically, CatB is a member of the cysteine cathepsin family that acts in cellular processes, such as tumor development and invasion. However, the relationship between CST1 and CatB, and their roles in tumor development are poorly understood. In this study, we observed that the knockdown of CST1 induced the activity of senescence-associated ß-galactosidase, a marker of cellular senescence, and expression of senescence-associated secretory phenotype genes, including interleukin-6 and chemokine (C-C motif) ligand 20, in MDA-MB-231 and SW480 cancer cells. Furthermore, CST1 knockdown decreased extracellular CatB activity, and direct CatB inhibition, using specific inhibitors or shCatB, induced cellular senescence. Reconstitution of CST1 restored CatB activity and inhibited cellular senescence in CST1 knockdown cells. CST1 knockdown or CatB inhibition increased glycogen synthase (GS) kinase 3ß phosphorylation at serine 9, resulting in the activation of GS and the induction of glycogen accumulation associated with cellular senescence. Importantly, CST1 knockdown suppressed cancer cell proliferation, soft agar colony growth and tumor growth in a xenograft model. These results indicate that CST1-mediated extracellular CatB activity enhances tumor development by preventing cellular senescence. Our findings suggest that antagonists of CST1 or inhibitors of CatB are potential anticancer agents.
Assuntos
Catepsina B/metabolismo , Proliferação de Células , Glicogênio Sintase/metabolismo , Glicogênio/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Cistatinas Salivares/metabolismo , Animais , Catepsina B/genética , Senescência Celular/genética , Técnicas de Silenciamento de Genes , Glicogênio/genética , Glicogênio Sintase/genética , Células HEK293 , Xenoenxertos , Humanos , Células MCF-7 , Camundongos , Proteínas de Neoplasias/genética , Transplante de Neoplasias , Neoplasias/genética , Cistatinas Salivares/genéticaRESUMO
A major goal of breast cancer research is to prevent the molecular events that lead to tumour metastasis. It is well-established that both cytoplasmic and mitochondrial reactive oxygen species (ROS) play important roles in cell migration and metastasis. Accordingly, this study examined the molecular mechanisms of the anti-metastatic effects of NecroX-5, a mitochondrial ROS scavenger. NecroX-5 inhibited lung cancer metastasis by ameliorating migration in a mouse model. In human cancer cells, the inhibition of migration by NecroX-5 is cell type-dependent. We observed that the effect of NecroX-5 correlated with a reduction in mitochondrial ROS, but mitochondrial ROS reduction by MitoQ did not inhibit cell migration. NecroX-5 decreased intracellular calcium concentration by blocking Ca2+ influx, which mediated the inhibition of cell migration, AKT downregulation and the reduction of mitochondrial ROS levels. However, the reduction of mitochondrial ROS was not associated with supressed migration and AKT downregulation. Our study demonstrates the potential of NecroX-5 as an inhibitor of breast cancer metastasis.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Movimento Celular/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/administração & dosagem , Proteína Oncogênica v-akt/biossíntese , Sulfonas/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Cálcio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Metástase Neoplásica , Proteína Oncogênica v-akt/genética , Compostos Organofosforados/administração & dosagem , Espécies Reativas de Oxigênio/metabolismo , Ubiquinona/administração & dosagem , Ubiquinona/análogos & derivados , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Hepatitis C virus (HCV) E2 protein binds to CD81, which is a component of the B cell co-stimulatory complex. The E2-CD81 interaction leads to B cell proliferation, protein tyrosine phosphorylation and to the hypermutation of immunoglobulin genes. Epidemiological studies have reported a high prevalence of B cell non-Hodgkin lymphoma (NHL) in HCV-positive patients, suggesting a potential association between HCV and Epstein-Barr virus (EBV) in the genesis of B lymphocyte proliferative disorders. In the present study, in order to investigate the association between EBV and HCV in B cells, we created an in vitro EBV-induced B cell transformation model. CD81 was gradually overexpressed during transformation by EBV. B cells isolated from HCV-positive patients grew more rapidly and clumped together earlier than B cells isolated from healthy donors following EBV infection. Pre-stimulation of CD81 expressed by resting B cells with anti-CD81 monoclonal antibody (mAb) or HCV E2 accelerated the generation of lymphoblastoid cell lines (LCLs) by EBV infection. These cells proliferated prominently through the early expression of interleukin-10 and intracellular latent membrane protein (LMP)-l. By contrast, the overexpression of CD81 on EBV-transformed B cells by anti-CD81 mAb or HCV E2 protein induced apoptosis through reactive oxygen species (ROS)-mediated mitochondrial dysfunction. These results suggest that the engagement of CD81 expressed by B cells has differential effects on B cell fate (proliferation or apoptosis) according to EBV infection and the expression level of CD81.
Assuntos
Apoptose/imunologia , Linfócitos B/imunologia , Proliferação de Células , Transformação Celular Viral/imunologia , Herpesvirus Humano 4/imunologia , Tetraspanina 28/imunologia , Adulto , Anticorpos/imunologia , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Linfócitos B/metabolismo , Linfócitos B/virologia , Western Blotting , Células Cultivadas , Infecções por Vírus Epstein-Barr/imunologia , Infecções por Vírus Epstein-Barr/metabolismo , Infecções por Vírus Epstein-Barr/virologia , Feminino , Hepacivirus/imunologia , Hepacivirus/fisiologia , Hepatite C/imunologia , Hepatite C/metabolismo , Hepatite C/virologia , Herpesvirus Humano 4/fisiologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Masculino , Potencial da Membrana Mitocondrial/imunologia , Microscopia Confocal , Pessoa de Meia-Idade , Ligação Proteica , Espécies Reativas de Oxigênio/imunologia , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraspanina 28/metabolismo , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/metabolismoRESUMO
Our previous study demonstrated that G-CSF treatment increased the expression of TLR2 in donor grafts; this contributed to rapid engraftment after allogeneic hematopoietic stem cell transplantation (HSCT) in mice. In the current study, we investigated the effects of upregulated TLR2 expression in G-CSF-mobilized donor grafts on acute graft-versus-host disease (GVHD). We found that TLR2 was highly expressed on myeloid cell populations but not T and B cells from the spleens of G-CSF-treated donor mice. After transplantation, the mortality and disease severity in recipients were not significantly different between G-CSF-treated TLR2-/- and wt donor grafts. Although endogenous TLR2 ligand was detected in the serum of both recipients, T cells from TLR2-/- and wt donors have the same ability regarding alloreactivity. Moreover, the blockade of TLR2 signaling in recipients by administering anti-TLR2 blocking antibody after BMT did not lead to a significant difference in acute GVHD compared with control IgG treatment. However, the hematopoietic ability of G-CSF-mobilized lin−c-kit+ HSCs from TLR2-/- donor grafts was lower than that from wt donor grafts. Our results demonstrate that upregulated TLR2 expression in G-CSF-mobilized donor grafts has no effect on acute GVHD, suggesting that TLR2 is a valuable target for increasing HSCT efficiency in order to enhance engraftment without exacerbating acute GVHD.
Assuntos
Doença Enxerto-Hospedeiro/imunologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Receptor 4 Toll-Like/biossíntese , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Transplante de Células-Tronco Hematopoéticas/métodos , Camundongos , Camundongos Knockout , Células Mieloides/efeitos dos fármacos , Células Mieloides/metabolismo , Transplante de Células-Tronco , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Receptor 4 Toll-Like/genéticaRESUMO
Aberrant B7-H4 expression in cancer tissues serves as a novel prognostic biomarker for poor survival in patients with cancer. However, the factor(s) that induce cancer cell-associated B7-H4 remain to be fully elucidated. We herein demonstrate that hypoxia upregulates B7-H4 transcription in primary CD138(+) multiple myeloma cells and cancer cell lines. In support of this finding, analysis of the Multiple Myeloma Genomics Portal (MMGP) data set revealed a positive correlation between the mRNA expression levels of B7-H4 and the endogenous hypoxia marker carbonic anhydrogenase 9. Hypoxia-induced B7-H4 expression was detected in the cytoplasm, but not in cancer cell membranes. Chromatin immunoprecipitation analysis demonstrated binding of hypoxia-inducible factor-1α (HIF-1α) to proximal hypoxia-response element (HRE) sites within the B7-H4 promoter. Knockdown of HIF-1α and pharmacological inhibition of HIF-1α diminished B7-H4 expression. Furthermore, knockdown of cytoplasmic B7-H4 in MCF-7 decreased the S-phase cell population under hypoxia. Finally, MMGP analysis revealed a positive correlation between the transcript levels of B7-H4 and proliferation-related genes including MKI67, CCNA1, and Myc in several patients with multiple myeloma. Our results provide insight into the mechanisms underlying B7-H4 upregulation and its role in cancer cell proliferation in a hypoxic tumor microenvironment.
Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Mieloma Múltiplo/metabolismo , Inibidor 1 da Ativação de Células T com Domínio V-Set/biossíntese , Hipóxia Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Imunoprecipitação da Cromatina , Citoplasma/metabolismo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células MCF-7 , Mieloma Múltiplo/genética , Mieloma Múltiplo/patologia , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/fisiologia , Inibidor 1 da Ativação de Células T com Domínio V-Set/antagonistas & inibidores , Inibidor 1 da Ativação de Células T com Domínio V-Set/genéticaRESUMO
PURPOSE: Platelet-activating factor (PAF) has been found in various ocular tissues; the activity of PAF depends on the binding to its specific receptor, PAF-receptor. We investigated the therapeutic effects of PAF-receptor antagonists (CV-3988 and Ginkgolide B) on alkali burn-induced corneal neovascularization (CNV). METHODS: CNV was induced by applying a 0.2 N sodium hydroxide (3 µl, NaOH) solution directly on mice corneas. CV-3988 (1 mM/10 µl) and Ginkgolide B (1 mM/10 µl) were administered topically on the corneas three times daily for three consecutive days. CNV was evaluated under a slit-lamp microscope. Corneas were processed for histological, immunohistochemical and reverse transcription polymerase chain reaction analysis. Human umbilical vein endothelial cells were used for the migration and tube formation assay. RESULTS: Application of CV-3988 and Ginkgolide B inhibited CNV caused by alkali burn. CV-3988 and Ginkgolide B attenuated the expression of PAF-receptor mRNA. Alkali injury induced a massively increased intraocular mRNA expression of an angiogenic factor in cornea tissues, whereas these increments were attenuated by the application of CV-3988 and Ginkgolide B. CONCLUSIONS: CV-3988 and Ginkgolide B reversed opacity and neovascularization in alkali burn-induced corneas. Our findings suggest that CV-3988 and Ginkgolide B may be therapeutically useful in the treatment of CNV and inflammation.
Assuntos
Neovascularização da Córnea/tratamento farmacológico , Queimaduras Oculares/tratamento farmacológico , Ginkgolídeos/uso terapêutico , Lactonas/uso terapêutico , Éteres Fosfolipídicos/uso terapêutico , Inibidores da Agregação Plaquetária/uso terapêutico , Glicoproteínas da Membrana de Plaquetas/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Álcalis/efeitos adversos , Animais , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Lesões da Córnea/induzido quimicamente , Neovascularização da Córnea/patologia , Opacidade da Córnea/tratamento farmacológico , Queimaduras Oculares/induzido quimicamente , Queimaduras Oculares/patologia , Feminino , Ginkgolídeos/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Lactonas/farmacologia , Camundongos , Neovascularização Fisiológica/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Éteres Fosfolipídicos/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Glicoproteínas da Membrana de Plaquetas/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genéticaRESUMO
Numerous treatments have been used in the management of corneal chemical burns; however, no optimal treatment for corneal chemical burns currently exists. The present study investigated the effects of topical chondrocyte-derived extracellular matrix (CD-ECM) treatment on corneal wound healing, using an alkali burn mouse model. Topical treatment with CD-ECM was shown to reduce corneal opacity following an alkali burn. A histological examination observed the presence of regenerated epithelial cells and a small number of inflammatory cells in the corneas of CD-ECM-treated mice. The majority of the inflammatory cells present in the corneas of the phosphate-buffered saline (PBS)-treated mice were neutrophils that expressed matrix metalloproteinase (MMP)-9. The amount of neutrophils was significantly reduced in the corneas of the CD-ECM-treated mice. Furthermore, the expression levels of interleukin (IL)-8 were significantly reduced in the CD-ECM treatment group, but not in the mice that received the PBS treatment. The results of the present study indicate that CD-ECM treatment may accelerate wound healing in a model of alkali burn-induced corneal injury. The therapeutic mechanism may be associated with accelerated reepithelialization and reduced recruitment of MMP-9-expressing neutrophils, through inhibiting the production of IL-8.
Assuntos
Queimaduras Químicas , Condrócitos , Lesões da Córnea/induzido quimicamente , Matriz Extracelular , Queimaduras Oculares/induzido quimicamente , Cicatrização/efeitos dos fármacos , Administração Tópica , Animais , Lesões da Córnea/tratamento farmacológico , Modelos Animais de Doenças , Queimaduras Oculares/tratamento farmacológico , Feminino , Interleucina-8/biossíntese , Metaloproteinase 9 da Matriz , Camundongos , Infiltração de Neutrófilos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologiaRESUMO
CONTEXT: Expression of various inflammatory mediators in corneal fibroblasts contributes to corneal inflammation. OBJECTIVE: The purpose of this study was to assess the possible effects of caffeic acid phenethyl ester (CAPE) on the expression of inflammatory mediators during an inflammatory response in human corneal fibroblasts. MATERIALS AND METHODS: The levels of interleukin (IL)-6, monocyte chemotactic protein (MCP)-1, and intercellular adhesion molecule-1 (ICAM-1) from IL-1ß-exposed human corneal fibroblasts were measured with enzyme-linked immunosorbent assays (ELISA). The regulatory mechanisms of CAPE on cellular signaling pathways were examined using Western blot and electrophoretic mobility shift assays. A functional validation was carried out by evaluating the inhibitory effects of CAPE on neutrophil and monocyte migration in vitro. RESULTS: CAPE inhibited the expression of IL-6, MCP-1 and ICAM-1 induced by the pro-inflammatory cytokine IL-1ß in corneal fibroblasts. The activation of AKT and NF-κB by IL-1ß was markedly inhibited by CAPE, whereas the activity of mitogen-activated protein kinases (MAPKs) was not affected. CAPE significantly suppressed the IL-1ß-induced migration of differentiated (d)HL-60 and THP-1 cells. DISCUSSION: These anti-inflammatory effects of CAPE may be expected to inhibit the infiltration of leukocytes into the corneal stroma in vivo.
Assuntos
Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Ácidos Cafeicos/farmacologia , Córnea/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Interleucina-1beta/antagonistas & inibidores , Interleucina-1beta/farmacologia , Álcool Feniletílico/análogos & derivados , Adulto , Moléculas de Adesão Celular/efeitos dos fármacos , Ensaios de Migração de Leucócitos , Movimento Celular/efeitos dos fármacos , Quimiocinas/efeitos dos fármacos , Córnea/citologia , Citocinas/efeitos dos fármacos , Células HL-60 , Humanos , Pessoa de Meia-Idade , Álcool Feniletílico/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
AIM: B7-H1, which belongs to the B7 family of costimulatory molecules, is implicated in the ability of tumors to evade the host immune response. The development of evasion mechanisms within the tumor microenvironment may be responsible for poor therapeutic responses. In this manuscript, we report that the 15-deoxy-δ(12,14)-prostaglandin J2 (15d-PGJ2), peroxisome proliferator-activated receptor gamma (PPARγ) activator leads to the downregulation of the cancer-associated expression of B7-H1 in response to interferon-gamma (IFN-γ) and the associated signaling cascades. MAIN METHODS: The expression of B7-H1 from IFN-γ-induced B16F10 melanoma cells was measured with flow cytometric analysis. The regulatory mechanisms of 15d-PGJ2 on cellular signaling pathways were examined using Western blot and electrophoretic mobility shift assays. KEY FINDINGS: The flow cytometric analysis revealed that the B7-H1 costimulatory molecule is significantly upregulated in B16F10 melanoma cells by stimulation with IFN-γ. However, 15d-PGJ2 strongly downregulates B7-H1 expression in IFN-γ-stimulated B16F10 melanoma cells. Furthermore, the significant damping effect of 15d-PGJ2 on B7-H1 expression involves the inhibition of the tyrosine phosphorylation of Janus kinase (Jak) and signal transducer(s) and activator(s) of transcription (STAT) and, thereby, the interferon regulatory factor-1 (IRF-1) trans-activation of STAT. These effects of 15d-PGJ2 were not abrogated by the PPARγ antagonist GW9662, indicating that they occur through a PPARγ-independent mechanism. SIGNIFICANCE: In this study, we demonstrate that 15d-PGJ2 suppresses the IFN-γ-elicited expression of B7-H1 by the inhibition of IRF-1 transcription via the Jak/STAT signaling pathway through a PPARγ-independent mechanism in mouse melanoma cells.
Assuntos
Antígeno B7-H1/genética , Regulação Neoplásica da Expressão Gênica , Interferon gama/farmacologia , Prostaglandina D2/análogos & derivados , Transdução de Sinais/efeitos dos fármacos , Anilidas/farmacologia , Animais , Antígeno B7-H1/metabolismo , Linhagem Celular Tumoral , Fator Regulador 1 de Interferon/antagonistas & inibidores , Fator Regulador 1 de Interferon/genética , Fator Regulador 1 de Interferon/metabolismo , Janus Quinases/antagonistas & inibidores , Janus Quinases/genética , Janus Quinases/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Prostaglandina D2/farmacologia , Fatores de Transcrição STAT/antagonistas & inibidores , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismoRESUMO
PURPOSE: To investigate the effect of the overexpression of miRNA-9 to the ratio of pro- and anti-angiogenic isoforms of vascular endothelial growth factor (VEGF) in human retinal pigment cells (ARPE-19). METHODS: Oxidative stress was induced to ARPE-19 cells by 4-hydroxynonenal (4-HNE), tert-butyl hydroperoxide (t-BH), and hypoxia chamber with 1% O2. Expression patterns of miRNAs were validated by qPCR. Relative mRNA levels of VEGF and PEDF were measured by semi-quantitative PCR. After the transfection of miR-9 mimic and inhibitor, transcriptional levels of VEGF165a, VEGF 165b, and SRPK-1 were measured by qPCR. RESULTS: We demonstrated that miR-9 expression is decreased in ARPE-19 human retinal pigment cells under hypoxic stress induced by 4-HNE, a lipid peroxidation end-product. We observed that miR-9 mimic transfection of ARPE-19 inhibited one of its targets, serine-arginine protein kinase-1 (SRPK-1), modulating the transcriptional level of VEGF165b. Transfection of miR-9 reduced the alternative splicing of VEGF165a mRNA in ARPE-19 cells under hypoxic conditions, suggesting that miR-mediated regulation of alternative splicing could be a potential therapeutic target in neovascular pathologies. CONCLUSIONS: Hypoxic stress decreased the miR-9 level in ARPE-19 cells, which increased the transcriptional level of SRPK-1, resulting in alternative splicing shift to pro-angiogenic isoforms of VEGF165 in human retinal pigment epithelial cells.
Assuntos
Regulação da Expressão Gênica/fisiologia , MicroRNAs/genética , Estresse Oxidativo , Proteínas Serina-Treonina Quinases/genética , Epitélio Pigmentado da Retina/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Aldeídos/toxicidade , Linhagem Celular , Relação Dose-Resposta a Droga , Proteínas do Olho/genética , Humanos , Immunoblotting , Fatores de Crescimento Neural/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Epitélio Pigmentado da Retina/efeitos dos fármacos , Serpinas/genética , Transfecção , terc-Butil Hidroperóxido/toxicidadeRESUMO
CONTEXT: Caffeic acid phenethyl ester (CAPE), an active component of honeybee propolis, is known to have antioxidant, anti-inflammatory, and other beneficial medicinal properties. However, the molecular mechanisms underlying its anti-allergic effects in mast cells are unknown. OBJECTIVE: The purpose of the present study was to examine whether CAPE modulates the immunoglobulin E (IgE)-mediated local allergic reaction in animals, as well as to elucidate the effects of CAPE on mast cells in vitro. MATERIALS AND METHODS: To investigate the bioactive potential of CAPE (10 or 20 µM), HMC-1 cells were stimulated with phorbol 12-myristate 13-acetate plus calcium ionophore A23187 (PMACI) for 24 h in the presence or absence of CAPE. To study the pharmacological effects of CAPE, enzyme-linked immunosorbent assays (ELISAs), RT-PCR, Western blot analysis, electrophoretic mobility shift assays (EMSAs), and fluorescence assays were used. RESULTS: CAPE (10 mg/kg) inhibited local IgE-mediated allergic reactions (0.164 versus 0.065 O.D.) in a mouse model. Additionally, CAPE (20 µM) attenuated PMACI-stimulated histamine release (3146.42 versus 2564.83 pg/ml) and the production of inflammatory cytokines, such as interleukin (IL)-1ß (4.775 versus 0.713 pg/ml, IC50 = 6.67 µM), IL-6 (4771.5 versus 449.1 pg/ml, IC50 = 5.25 µM), and IL-8 (5991.7 versus 2213.1 pg/ml, IC50 = 9.95 µM) in HMC-1 cells. In activated HMC-1 cells, pretreatment with CAPE decreased the phosphorylation of c-Jun N-terminal kinase. In addition, CAPE inhibited PMACI-induced nuclear factor (NF)-κB activation by suppressing IκBα phosphorylation and its degradation. DISCUSSION AND CONCLUSION: Our results indicated that CAPE can modulate mast cell-mediated allergic disease.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Ácidos Cafeicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Álcool Feniletílico/análogos & derivados , Animais , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/efeitos dos fármacos , Histamina/metabolismo , Liberação de Histamina/efeitos dos fármacos , Humanos , Hipersensibilidade/prevenção & controle , Imunoglobulina E/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Mastócitos/citologia , Mastócitos/metabolismo , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Álcool Feniletílico/farmacologia , Fosforilação/efeitos dos fármacosRESUMO
Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease. Gallium nitrate has been reported to reserve immunosuppressive activities. Therefore, we assessed the therapeutic effects of gallium nitrate in the mouse model of developed type II collagen-induced arthritis (CIA). CIA was induced by bovine type II collagen with Complete Freund's adjuvant. CIA mice were intraperitoneally treated from day 36 to day 49 after immunization with 3.5mg/kg/day, 7mg/kg/day gallium nitrate or vehicle. Gallium nitrate ameliorated the progression of mice with CIA. The clinical symptoms of collagen-induced arthritis did not progress after treatment with gallium nitrate. Gallium nitrate inhibited the increase of CD4(+) T cell populations (p<0.05) and also inhibited the type II collagen-specific IgG2a-isotype autoantibodies (p<0.05). Gallium nitrate reduced the serum levels of TNF-α, IL-6 and IFN-γ (p<0.05) and the mRNA expression levels of these cytokine and MMPs (MMP2 and MMP9) in joint tissues. Western blotting of members of the NF-κB signaling pathway revealed that gallium nitrate inhibits the activation of NF-κB by blocking IκB degradation. These data suggest that gallium nitrate is a potential therapeutic agent for autoimmune inflammatory arthritis through its inhibition of the NF-κB pathway, and these results may help to elucidate gallium nitrate-mediated mechanisms of immunosuppression in patients with RA.
Assuntos
Anti-Inflamatórios/uso terapêutico , Artrite Experimental/tratamento farmacológico , Gálio/uso terapêutico , Animais , Articulação do Tornozelo/efeitos dos fármacos , Articulação do Tornozelo/metabolismo , Articulação do Tornozelo/patologia , Anti-Inflamatórios/farmacologia , Artrite Experimental/induzido quimicamente , Artrite Experimental/imunologia , Artrite Experimental/patologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Colágeno Tipo II , Pé/patologia , Gálio/farmacologia , Imunoglobulina G/sangue , Interferon gama/sangue , Interferon gama/genética , Interleucina-6/sangue , Interleucina-6/genética , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Camundongos Endogâmicos DBA , NF-kappa B/antagonistas & inibidores , NF-kappa B/imunologia , Baço/citologia , Baço/efeitos dos fármacos , Baço/imunologia , Fator de Necrose Tumoral alfa/sangue , Fator de Necrose Tumoral alfa/genéticaRESUMO
A well-recognized natural ligand of PPARγ, 15-deoxy-δ(12,14)-prostaglandin J(2) (15d-PGJ(2)) possesses immunomodulatory properties. The aim of this study was to elucidate whether 15d-PGJ(2) was able to attenuate lipopolysaccharide (LPS)-induced inflammatory responses in human retinal pigment epithelial (RPE) cells, which are involved in ocular immune responses. In addition, we examined whether the platelet activating factor (PAF) is associated with the anti-inflammatory activity of 15d-PGJ(2). ARPE19 cells treated with varying concentrations of 15d-PGJ(2) and a PAF antagonist (CV3988) were used in this study. The activity of PAF-acetylhydrolase (PAF-AH) was assayed by treatment with 15d-PGJ(2) and CV3988 in the presence of LPS. 15d-PGJ(2) and CV3988 inhibited the LPS-induced mRNA expression and protein production of interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and intercellular adhesion molecule-1 (ICAM-1) in ARPE19 cells. These effects resulting from 15d-PGJ(2) were not abrogated by the PPARγ antagonist, indicating that the actions were PPARγ-independent. Furthermore, 15d-PGJ(2) and CV3988 enhanced the PAF-AH activity. Additionally, 15d-PGJ(2) inhibited the phosphorylation of the extracellular signal-regulated kinase (ERK) and the activation of nuclear transcription factor-κB (NF-κB). These results demonstrated that 15d-PGJ(2) reduced LPS-stimulated inflammatory responses in ARPE19 cells by enhancing the PAH-AH activity. These results suggest that 15d-PGJ(2) may have potent anti-inflammatory activity against ocular inflammation.
Assuntos
1-Alquil-2-acetilglicerofosfocolina Esterase/metabolismo , Anti-Inflamatórios/farmacologia , Células Epiteliais/enzimologia , Inflamação/metabolismo , Prostaglandina D2/análogos & derivados , Sobrevivência Celular/efeitos dos fármacos , Quimiocina CCL2/antagonistas & inibidores , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Inflamação/induzido quimicamente , Molécula 1 de Adesão Intercelular/genética , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-6/antagonistas & inibidores , Interleucina-6/genética , Interleucina-6/metabolismo , Lipopolissacarídeos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , NF-kappa B/metabolismo , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Fosforilação , Prostaglandina D2/farmacologia , Epitélio Pigmentado da Retina/citologiaRESUMO
The generation of tryptophan (Trp) metabolites by indoleamine 2,3-dioxygenase (IDO) is an effective mechanism for T cell suppression. However, the effect of Trp metabolites on dendritic cells (DCs) remains unclear. Here, we investigated whether the tryptophan metabolite 3-hydroxyanthranilic acid (3-HAA) directly inhibits DC activation and is responsible for T cell suppression. We found that 3-HAA treatment significantly reduced IL-12, IL-6, and TNF-α production in bone marrow-derived DCs (BMDCs) stimulated with LPS. Maturation markers CD40, CD80, CD86, and I-A were also significantly reduced. Moreover, treatment with 3-HAA decreased the ability of DCs to stimulate T cell activation and differentiation in vitro and in vivo. Finally, we observed that phospho-JNK and phospho-38 levels were reduced in 3-HAA-treated DC2.4 cells and BMDCs. These results suggest that the tryptophan metabolite 3-HAA suppresses T cell responses by inhibiting DC activation.