RESUMO
A high-fat/high-cholesterol (HFC) diet, but not a high-cholesterol (HC) diet, is known to induce elevated serum apolipoprotein E (apoE)-rich high-density lipoprotein (HDL) levels in animal models. However, the exact mechanisms by which the combination of dietary fat and cholesterol induces apoE-rich HDL production is not well understood. Here, we investigated the effects of dietary fat and cholesterol on serum lipoprotein profiles and hepatic mRNA expression that are associated with HDL production, cholesterol transport, and bile acid metabolism. Male Sprague-Dawley rats were fed HFC, HC, high-fat, or control diets and then evaluated. The HFC diet induced significant increases in hepatic free-cholesterol accumulation (1.4-fold, p < 0.01) and serum apoE-rich HDL cholesterol (8.7-fold, p < 0.001) levels compared with the HC diet. The apoE-rich HDL induced by the HFC diet was remarkably rich in free cholesterol. Liver gene-expression was mostly similar between the HC and HFC diet groups. However, there was a significant increase of ATP-binding cassette transporter A1 (ABCA1) levels in the HFC group compared to the HC group for both mRNA (1.9-fold, p < 0.001) and protein (6.6-fold, p < 0.01). These results suggest that an increase in apoE-rich HDL induced by dietary fat and cholesterol may be involved in cholesterol efflux from the liver through increased ABCA1-mediated free-cholesterol efflux.
Assuntos
Transportador 1 de Cassete de Ligação de ATP , Colesterol , Dieta Hiperlipídica , Transportador 1 de Cassete de Ligação de ATP/genética , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP , Animais , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Colesterol/efeitos adversos , Colesterol/metabolismo , HDL-Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/metabolismo , Fígado/metabolismo , Masculino , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
A novel proteasome deubiquitinase inhibitor, VLX1570, has been highlighted as a promising therapeutic agent mainly for lymphoid neoplasms and solid tumors. We examined in vitro effects of VLX1570 on eight myeloid and three lymphoid leukemia cell lines. From cell culture studies, 10 out of 11 cell lines except K562 were found to be susceptible to VLX1570 treatment and it inhibited cell growth mainly by apoptosis. Next, to identify the signaling pathways associated with apoptosis, we performed gene expression profiling using HL-60 with or without 50 nmol/L of VLX1570 for 3 hours and demonstrated that VLX1570 induced the genetic pathway involved in "heat shock transcription factor 1 (HSF1) activation", "HSF1 dependent transactivation", and "Regulation of HSF1 mediated heat shock response". VLX1570 increased the amount of high molecular weight polyubiquitinated proteins and the expression of HSP70 as the result of the suppression of ubiquitin proteasome system, the expression of heme oxygenase-1, and the amount of phosphorylation in JNK and p38 associated with the generation of reactive oxygen species (ROS) induced apoptosis and the amount of phosphorylation in eIF2α, inducing the expression of ATF4 and endoplasmic reticulum (ER) stress dependent apoptosis protein, CHOP, and the amount of phosphorylation slightly in IRE1α, leading to increased expression of XBP-1s in leukemia cell lines. In the present study, we demonstrate that VLX1570 induces apoptosis and exerts a potential anti-leukemic effect through the generation of ROS and induction of ER stress in leukemia cell lines.
Assuntos
Antineoplásicos/farmacologia , Azepinas/farmacologia , Compostos de Benzilideno/farmacologia , Leucemia Linfoide/metabolismo , Leucemia Mieloide Aguda/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Células HL-60 , Humanos , Células K562 , Leucemia Linfoide/tratamento farmacológico , Leucemia Linfoide/genética , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genéticaRESUMO
BACKGROUND: High-density lipoprotein (HDL) containing apolipoprotein E (apoE-rich HDL) represents a small portion of plasma HDL. We recently established a method for measuring plasma apoE-rich HDL. This study aimed to investigate the relationship between metabolic syndrome (MetS) and apoE-rich HDL levels. METHODS: The apoE-rich HDL-cholesterol (HDL-C) levels and metabolic characteristics of 113 patients were analyzed. RESULTS: The MetS group (n = 58) had significantly lower apoE-rich HDL-C and a lower apoE-rich HDL-C/HDL-C ratio (apoE-HDL (%)) compared to the non-MetS group. The prevalence of MetS was increased when apoE-HDL (%) decreased. In simple regression analyses, apoE-HDL (%) was significantly inversely correlated with visceral fat area (rs = -0.370, P < 0.001) and plasma triglycerides (rs = -0.447, P < 0.001) and positively correlated with low-density lipoprotein (LDL) mean particle size (rs = 0.599, P < 0.001) and HDL mean particle size (rs = 0.512, P < 0.001). Stepwise multiple regression analysis revealed that LDL mean particle size, a component of the atherogenic lipoprotein profile, was an independent predictor of apoE-HDL (%) (adjusted R2 = 0.409). CONCLUSIONS: Plasma apoE-rich HDL levels might be a valuable indicator of MetS. These findings may help further understand HDL subfraction analysis in cardiometabolic diseases.
Assuntos
Síndrome Metabólica , Apolipoproteínas E , HDL-Colesterol , LDL-Colesterol , Humanos , Lipoproteínas HDL , TriglicerídeosRESUMO
To investigate megakaryocyte (MK) DNA ploidy in various hematological diseases, fluorescence microscopy imaging system (FMI) can be used to analyze DNA ploidy with cell morphology at the single-cell level by using specialized image-processing software. Here we compared DNA ploidy obtained by FMI measured with that obtained flow cytometry (FCM). With FMI, we could evaluate the DNA ploidy in long-term preserved bone marrow smear samples after staining. We next analyzed the MK DNA ploidy in 42 bone marrow smear samples including 26 myeloid neoplasm cases, and we compared the DNA ploidy and platelet counts in the patients' peripheral blood; the production of platelets was significantly high compared to DNA ploidy in the myeloproliferative neoplasms group. The FMI method revealed that the patients with 5q- syndrome exhibited relatively low DNA ploidy despite high platelet counts, and this result suggested that increased DNA ploidy is not indispensable to abundant platelet production. The FMI method for DNA ploidy will be a useful tool to clarify the relationship between DNA ploidy and platelet production by MKs.
Assuntos
Anemia Macrocítica/patologia , Megacariócitos/patologia , Microscopia de Fluorescência/métodos , Ploidias , Linhagem Celular Tumoral , Deleção Cromossômica , Cromossomos Humanos Par 5 , Citometria de Fluxo , HumanosRESUMO
Lavender essential oil (Lvn) has anti-inflammatory effects in an ovalbumin-sensitized murine model of asthma, and inhibits inflammatory cell infiltration into the lungs. The anti-inflammatory effects of Lvn on cell adhesion molecules are not clear. Here we evaluated the effects of Lvn and its main constituents, linalyl acetate (LA) and linalool (LO), on the expression of tumor necrosis factor-alpha (TNF-α)-induced cell adhesion molecules in murine brain endothelial bEnd.3 cells and human umbilical vein endothelial cells (HUVECs). The bEnd.3 cells were treated with Lvn, LA, or LO and subsequently stimulated with TNF-α. The mRNA expression levels of cell adhesion molecules were detected using RT-PCR. E-selectin and P-selectin protein and phosphorylated-NF-κB p65 were detected by western blotting. The effects of Lvn on HUVECs were measured by RT-PCR. In bEnd.3 cells, Lvn and LA suppressed TNF-α-induced E-selectin, P-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, and phosphorylated-NF-κB p65 in the nucleus; LO did not suppress P-selectin or phosphorylated-NF-κB p65. Lvn inhibited TNF-α-induced E-selectin mRNA in HUVECs. These results indicate that Lvn and LA inhibit TNF-α-induced cell adhesion molecules in endothelial cells through the suppression of NF-κB activation. Consequently, Lvn or other essential oils including LA may be useful as alternative anti-inflammatory medicines.
Assuntos
Moléculas de Adesão Celular/análise , Células Endoteliais/efeitos dos fármacos , Monoterpenos/farmacologia , Óleos Voláteis/farmacologia , Óleos de Plantas/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Monoterpenos Acíclicos , Animais , Moléculas de Adesão Celular/genética , Células Endoteliais/química , Células Endoteliais/metabolismo , Células Endoteliais da Veia Umbilical Humana/química , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Lavandula , Camundongos , NF-kappa B/fisiologia , Óleos Voláteis/análise , Óleos de Plantas/análise , Fator de Necrose Tumoral alfa/antagonistas & inibidoresRESUMO
AIMS: Lavender essential oil (Lvn) has been reported to have anti-inflammatory effects. Bronchial asthma is characterized by bronchial allergic inflammation with airway remodeling. Therefore, we evaluated the anti-inflammatory effect of Lvn on experimentally induced bronchial asthma in a murine model. MAIN METHODS: BALB/c mice were sensitized by an intraperitoneal injection of ovalbumin (OVA) at days 0 and 14, and subsequently challenged with nebulized OVA on days 28-30 (Control-Asthma group). Mice in the treatment group inhaled Lvn on days 14-31 (Lvn-Asthma group). The allergic inflammatory response was determined on days 32 and 33. KEY FINDINGS: An increase in airway resistance was inhibited in the Lvn-Asthma group than in the Control-Asthma group. The Lvn-Asthma group showed lower total cell numbers and eosinophils in bronchoalveolar lavage (BAL) fluids and peribronchial and perivascular tissues when compared with the Control-Asthma group. The Lvn-Asthma group also had less mucin hyperplasia than the Control-Asthma group. Furthermore, the Lvn-Asthma group showed lower interleukin (IL)-5 and IL-13 cytokine levels in BAL fluids, as well as reduced IL-4 and IL-5 mRNA expression in lung tissue, compared with the Control-Asthma group and determined by FlowCytomix and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. In addition, Lvn inhalation reduced Muc5b mRNA expression in the lungs without significantly changing the expression of Muc5ac mRNA. SIGNIFICANCE: Lvn inhibits allergic inflammation and mucous cell hyperplasia with suppression of T-helper-2 cell cytokines and Muc5b expression in a murine model of asthma. Consequently, Lvn may be useful as an alternative medicine for bronchial asthma.
Assuntos
Anti-Inflamatórios/farmacologia , Asma/tratamento farmacológico , Hiperplasia/tratamento farmacológico , Inflamação/tratamento farmacológico , Óleos Voláteis/farmacologia , Óleos de Plantas/farmacologia , Administração por Inalação , Resistência das Vias Respiratórias/efeitos dos fármacos , Animais , Anti-Inflamatórios/administração & dosagem , Asma/imunologia , Asma/patologia , Líquido da Lavagem Broncoalveolar/imunologia , Modelos Animais de Doenças , Feminino , Inflamação/imunologia , Inflamação/patologia , Interleucina-13/metabolismo , Interleucina-5/metabolismo , Lavandula , Camundongos , Camundongos Endogâmicos BALB C , Mucina-5B/genética , Óleos Voláteis/administração & dosagem , Ovalbumina/imunologia , Óleos de Plantas/administração & dosagem , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Th2/imunologia , Fatores de TempoRESUMO
The tyrosine kinase inhibitor (TKI) imatinib mesylate (IM) revolutionized the treatment of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph-ALL), which had showed poor prognosis before the dawn of IM treatment. However, if Ph-ALL patients showed IM resistance due to ABL kinase mutation, second-generation TKI, dasatinib or nilotinib, was recommended. We treated 4 pediatric Ph-ALL patients with both IM and bone marrow transplantation (BMT); however, 3 relapsed. We retrospectively examined the existence of ABL kinase mutation using PCR and direct sequencing methods, but there was no such mutation in all 4 diagnostic samples. Interestingly, two relapsed samples from patients who were not treated with IM before relapse did not show ABL kinase mutation and IM was still effective even after relapse. On the other hand, one patient who showed resistance to 3 TKI acquired dual ABL kinase mutations, F359C at the IM-resistant phase and F317I at the dasatinib-resistant phase, simultaneously. In summary, Ph-ALL patients relapsed with or without ABL kinase mutation. Furthermore, ABL kinase mutation was only found after IM treatment, so an IM-resistant clone might have been selected during the IM treatment and intensive chemotherapy. The appropriate combination of TKI and BMT must be discussed to cure Ph-ALL patients.
Assuntos
Proteínas de Fusão bcr-abl/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/diagnóstico , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Antineoplásicos/uso terapêutico , Medula Óssea/patologia , Criança , Pré-Escolar , Análise Mutacional de DNA , Humanos , Masculino , Recidiva Local de Neoplasia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêuticoRESUMO
AIMS: Fudosteine is a cysteine derivative that is used as an expectorant in chronic bronchial inflammatory disorders. It has been shown to decrease the number of goblet cells in an animal model. This study examined the effects of fudosteine on airway inflammation and remodeling in a murine model of chronic asthma. MAIN METHODS: BALB/c mice were sensitized by an intraperitoneal injection of ovalbumin (OVA), and subsequently challenged with nebulized ovalbumin three days a week for four weeks. Seventy-two hours after the fourth challenge, airway hyperresponsiveness (AHR) and the cell composition of bronchoalveolar lavage (BAL) fluid were assessed. Fudosteine was administered orally at 10mg/kg or 100mg/kg body weight from the first to the fourth challenge. KEY FINDINGS: We investigated the effects of fudosteine on the development of allergic airway inflammation and airway hyperresponsiveness after chronic allergen challenges. The administration of fudosteine during the challenge with ovalbumin prevented the development of airway hyperresponsiveness and accumulation of lymphocytes in the airways. Eotaxin, IL-4, and TGF-ß levels and the relative intensity of matrix metalloproteinase-2 and matrix metalloproteinase-9 (MMP-2 and MMP-9) in BAL fluid were reduced by the fudosteine treatment; however, the number of eosinophils in BAL fluid and serum IgE levels did not change. The expression of TGF-ß, the development of goblet cell hyperplasia, subepithelial collagenization, and basement membrane thickening were also reduced by the fudosteine treatment. SIGNIFICANCE: These results indicate that fudosteine is effective in reducing airway hyperresponsiveness, airway inflammation, and airway remodeling in a murine model of chronic asthma.
Assuntos
Hiper-Reatividade Brônquica , Cistina/análogos & derivados , Alérgenos/administração & dosagem , Animais , Líquido da Lavagem Broncoalveolar , Cistina/farmacologia , Feminino , Pulmão/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Fator de Crescimento Transformador beta/genéticaRESUMO
Dock2 has been shown to be indispensable for chemotaxis of mature lymphocytes as a critical Rac activator. However, the functional expression of Dock2 in immature hematopoietic cells is unclear. In this study, we demonstrate that Dock2 is broadly expressed in bone marrow (BM) hematopoietic compartment, including hematopoietic stem/progenitor cell (HSC/HPC) fraction. Response of Dock2-/- HPCs to CXCL12 in chemotaxis and actin polymerization in vitro was impaired, although alpha4 integrin activation by CXCL12 was not altered. Myelosuppressive stress on HSCs in vivo, such as consecutive 5-FU administration and serial bone marrow transplantation, did not show hematopoietic defect in Dock2-/- mice. Long-term engraftment of transplanted Dock2-/- BM cells was severely impaired in competitive reconstitution. However, this was not intrinsic to HSCs but originated from the defective competition of Dock2-/- lymphoid precursors. These results suggest that Dock2 plays a significant role in BM lymphopoiesis, but is dispensable for HSC engraftment and self-renewal.
Assuntos
Medula Óssea/efeitos dos fármacos , Proteínas Ativadoras de GTPase/farmacologia , Hematopoese/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Actinas/metabolismo , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Transplante de Medula Óssea/patologia , Quimiocina CXCL12/farmacologia , Quimiotaxia/fisiologia , Fluoruracila/farmacologia , Fatores de Troca do Nucleotídeo Guanina , Hematopoese/fisiologia , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Leucemia Mieloide/metabolismo , Leucemia Mieloide/patologia , Linfócitos/metabolismo , Linfócitos/patologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores CXCR4/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Células-Tronco/patologia , Fatores de TempoRESUMO
We previously demonstrated the simultaneous induction of urokinase-type plasminogen activator and interleukin-8, a CXC chemokine, in doxorubicin-treated human NCI-H69 small cell lung cancer cells in which extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase might be involved. NCI-H69 cells expressed one of the receptor tyrosine kinases, c-Kit, and STI571 inhibited the cell growth and stem cell factor-induced phosphorylation of c-Kit. We therefore investigated the effects of STI571 on the expression of urokinase-type plasminogen activator and interleukin-8 in NCI-H69 cells. Microarray analysis revealed the gene induction of not only urokinase-type plasminogen activator and interleukin-8, but also early growth response-1 in STI571-treated cells. Treatment with STI571 resulted in the induction of phosphorylation of all three mitogen-activated protein kinases, such as extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase and stress-activated protein kinase/c-jun N-terminal protein kinase. U0126, an inhibitor against extracellular signal-regulated kinase 1/2, however, only inhibited the STI571-induced interleukin-8 accumulation. Urokinase-type plasminogen activator and interleukin-8 are important biological factors in tumor cell regulation; STI571 may therefore influence many aspects of tumor cell biology through inducing urokinase-type plasminogen activator and interleukin-8, in which the induction of early growth response-1 expression and extracellular signal-regulated kinase 1/2 phosphorylation might be involved.
Assuntos
Carcinoma de Células Pequenas/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , Interleucina-8/biossíntese , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Pirimidinas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Benzamidas , Carcinoma de Células Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Mesilato de Imatinib , Análise de Sequência com Séries de Oligonucleotídeos , Piperazinas/uso terapêutico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Proto-Oncogênicas c-kit/metabolismo , Pirimidinas/uso terapêutico , Ativação TranscricionalRESUMO
We previously demonstrated the doxorubicin-induced expression of urokinase-type plasminogen activator (uPA), interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1) and tumor necrosis factor-alpha in human RC-K8 lymphoma cells and NCI-H69 small cell lung carcinoma cells in which reactive oxygen species might be involved. Amurubicin hydrochloride (AMR), a novel derivative drug of doxorubicin, was recently introduced to clinical practice for treatment of lung cancer in Japan. Therefore, we investigated the effects of AMR on the expression of uPA and chemokines in NCI-H69 cells. AMR and its active form, amurubicinol hydrochloride (AMROH), both induced the expression of uPA, IL-8 and MCP-1 in H69 cells in a dose-dependent manner. When the cultured supernatant obtained from AMR-treated H69 cells was subcutaneously injected into rabbits, migration of a significant number of eosinophils was observed around the injected site. Antigen levels of eotaxin-3, a major migration-factor of eosinophils, were increased in AMROH-treated cells in parallel with the mRNA levels. The induction was observed below the clinically achievable concentration of AMR or AMROH. Thus, the simultaneous induction of uPA, IL-8, MCP-1 and eotaxin-3 may play a role in the pharmacological action of AMR through induction of the interaction between proinflammatory cells and lung carcinoma cells.
Assuntos
Antraciclinas/farmacologia , Quimiocinas CC/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Animais , Northern Blotting , Carcinoma de Células Pequenas/genética , Carcinoma de Células Pequenas/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CCL26 , Quimiocinas CC/metabolismo , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Eosinófilos/citologia , Eosinófilos/efeitos dos fármacos , Eosinófilos/fisiologia , Humanos , Injeções Subcutâneas , Interleucina-8/genética , Interleucina-8/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Coelhos , Ativador de Plasminogênio Tipo Uroquinase/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
We previously reported the induction of interleukin-8 (IL-8), one of the CXC chemokines, by all-trans retinoic acid (ATRA) in PL-21 and NB4 human myeloid leukemia cells, which may be implicated in APL differentiation syndrome that is a relatively frequent complication in patients with acute promyelocytic leukemia (APL) during treatment with ATRA. We, therefore, further investigated the effects of ATRA on the expression of chemokine family in NB4 cells and APL cells prepared from two APL patients. The RNase protection assay using a multi-probe template set for human chemokines revealed that ATRA induced gene expressions of a number of CC chemokines, such as monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1alpha and MIP-1beta in NB4 cells. Their antigen levels were also increased in the cultured media. APL cells prepared from two APL patients showed gene expression of chemokines, such as IL-8, MCP-1, MIP-1alpha, and MIP-1beta when stimulated with ATRA in vitro. Furthermore, serum levels of IL-8, MIP-1beta and RANTES were increased during the course of ATRA treatment in both APL patients who developed APL differentiation syndrome. These chemokines are all chemoattractants of particular inflammatory cell types, including neutrophils, monocytes and lymphocytes; therefore, the simultaneous induction of these chemokines after stimulation with ATRA may exacerbate the hyper-inflammation observed in ATRA-induced APL differentiation syndrome.
Assuntos
Quimiocinas CC/genética , Quimiocinas CXC/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Antineoplásicos/farmacologia , Sequência de Bases , Northern Blotting , Linhagem Celular Tumoral , Primers do DNA , Humanos , Interleucina-8/biossíntese , Leucemia Promielocítica Aguda , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
To clarify the mechanism by which radon hot springs prevent cancer or not, in this study, blood was collected from residents in the Misasa hot spring district and in a control district. The level of a representative cancer-suppressive gene, p53, and the activity of a representative antioxidant enzyme, superoxide dismutase (SOD), were analyzed as indices. The level of serum p53 protein in the males in the Misasa hot spring district was found to be 2-fold higher than that in the control district, which is a significant difference. In the females in the Misasa hot spring district, SOD activity was approximately 15% higher than that in the control district, which is also statistically significant, and exceeded the reference range of SOD activity despite advanced age. These results suggested that routine exposure of the residents in the Misasa hot spring district to radon at a concentration about 3 times higher than the national mean induces trace active oxygen in vivo, potentiating products of cancer-suppressive gene and antioxidant function. As the p53 protein level was high in the residents in the Misasa hot spring district, apoptosis of cancer cells may readily occur.
Assuntos
Poluição do Ar em Ambientes Fechados/análise , Suscetibilidade a Doenças/sangue , Suscetibilidade a Doenças/epidemiologia , Regulação da Expressão Gênica/efeitos da radiação , Radônio/análise , Superóxido Dismutase/sangue , Proteína Supressora de Tumor p53/sangue , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/sangue , Feminino , Humanos , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Medição de Risco/métodos , Fatores de Risco , Estatística como AssuntoRESUMO
We previously demonstrated the doxorubicin-induced urokinase-type plasminogen activator (uPA) expression in human RC-K8 lymphoma cells and NCI-H69 small cell lung carcinoma cells in which reactive oxygen species might be involved. Western blotting analysis revealed phosphorylation/activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) 1/2, p38 MAP kinase and stress-activated protein kinase/c-jun N-terminal protein kinase (SAPK/JNK) in doxorubicin-treated RC-K8 and H69 cells, and, therefore, we attempted to identify the MAP kinases implicated in doxorubicin-induced uPA expression by the use of their specific inhibitors. U0126, SB202190 and JNKI-1, inhibitors for MAPK kinase, (MEK) 1/2, p38 MAP kinase and SAPK/JNK, respectively, specifically and clearly inhibited their corresponding kinases. U0126 and SB202190, but not JNKI-1, almost completely inhibited the doxorubicin-induced uPA expression in both RC-K8 and H69 cells. However, U0126 rather enhanced the doxorubicin-induced activation of caspase-3 and poly ADP-ribose polymerase (PARP), and U0126 itself activated caspase-3 and PARP. Interestingly, JNKI-1 inhibited the doxorubicin-induced activation of caspase-3 and PARP. Therefore, doxorubicin treatment activates the above three kinases, but different MAP kinase signaling is responsible in the doxorubicin-induced caspase activation and expression of uPA. Thus, we could possibly manipulate the direction of doxorubicin-induced MAP kinase activation and the effects of doxorubicin on the tumor cell biology by the use of MAP kinase inhibitors.
Assuntos
Carcinoma de Células Pequenas/metabolismo , Doxorrubicina/farmacologia , Linfoma/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Northern Blotting , Western Blotting , Butadienos/farmacologia , Carcinoma de Células Pequenas/enzimologia , Caspase 3 , Caspases/metabolismo , Linhagem Celular Tumoral , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imidazóis/farmacologia , Neoplasias Pulmonares/metabolismo , Linfoma/enzimologia , MAP Quinase Quinase 4/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Nitrilas/farmacologia , Fosforilação , Poli Adenosina Difosfato Ribose/metabolismo , Piridinas/farmacologia , Ativador de Plasminogênio Tipo Uroquinase/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidoresAssuntos
Amoxicilina/administração & dosagem , Antibacterianos/administração & dosagem , Claritromicina/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Gastrite/tratamento farmacológico , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori , Omeprazol/análogos & derivados , Omeprazol/administração & dosagem , Trombocitopenia , 2-Piridinilmetilsulfinilbenzimidazóis , Idoso , Quimioterapia Combinada , Feminino , Gastrite/complicações , Gastrite/microbiologia , Gastrite/patologia , Infecções por Helicobacter/complicações , Infecções por Helicobacter/patologia , Humanos , Lansoprazol , Contagem de Plaquetas , Indução de Remissão , Trombocitopenia/complicações , Trombocitopenia/patologiaAssuntos
Antibacterianos/uso terapêutico , Infecções por Helicobacter/tratamento farmacológico , Helicobacter pylori , Minociclina/uso terapêutico , Ofloxacino/uso terapêutico , Omeprazol/análogos & derivados , Omeprazol/uso terapêutico , Púrpura Trombocitopênica Idiopática/tratamento farmacológico , 2-Piridinilmetilsulfinilbenzimidazóis , Plaquetas/efeitos dos fármacos , Quimioterapia Combinada , Infecções por Helicobacter/sangue , Humanos , Lansoprazol , Masculino , Pessoa de Meia-Idade , Contagem de Plaquetas , Púrpura Trombocitopênica Idiopática/sangue , Púrpura Trombocitopênica Idiopática/microbiologiaRESUMO
PURPOSE: We have previously demonstrated doxorubicin-induced urokinase (uPA) and interleukin-8 (IL-8) expression in human H69 small-cell lung carcinoma (SCLC) cells by a microarray technique using Human Cancer Chip version 2, in which 425 human "cancer-related" genes are spotted on the plates. The microarray analysis also revealed a significant induction of tumor necrosis factor-alpha (TNF-alpha), and doxorubicin-induced macrophage chemoattractant protein-1 (MCP-1) expression was demonstrated by an RNase protection assay. We extended the study by testing the effects of doxorubicin on the induction of TNF-alpha, uPA, IL-8 and MCP-1 in other types of lung carcinoma cells. METHODS: We investigated the effects of doxorubicin on the expression of TNF-alpha, uPA, IL-8 and MCP-1 in 12 human lung carcinoma cell lines, including five SCLC, three adenocarcinoma and four squamous cell carcinoma cells. The surface expression of their receptors was also investigated. RESULTS: TNF-alpha was significantly induced in three cell lines, H69, SBC-7 (SCLC) and PC-9 (adenocarcinoma), uPA in five cell lines, H69, SBC-7, EBC-1 (squamous cell), EBC-2 (squamous cell), and Sq-1 (squamous cell), IL-8 in three cell lines, H69, PC-9 and EBC-1, and MCP-1 in five cell lines, H69, SBC-3 (SCLC), SBC-7, PC-9 and Sq-1. In H69 cells, TNF-alpha antigen levels were increased approximately fivefold in the conditioned medium of doxorubicin-treated cells, in parallel with an increase in mRNA levels. As with uPA and IL-8, the maximum induction was observed at the "sublethal" concentrations of 2 and 4 microM at which cell growth was slightly inhibited 24 h after treatment. Furthermore, the cells did not express receptors including types I and II TNF-alpha receptors, uPA receptor (uPAR), C-x-C-chemokine receptor-1 (CXCR-1), or C-C-chemokine receptor-2, corresponding to TNF-alpha, uPA, IL-8 and MCP-1, respectively, that were induced by doxorubicin in the cells, although SBC-7 cells expressed uPAR, and EBC-1 cells expressed CXCR-1. CONCLUSIONS: TNF-alpha, uPA, IL-8 and MCP-1 induced and secreted from tumor cells upon doxorubicin stimulation may activate surrounding cells expressing the receptors such as neutrophils and monocytes/macrophages in a paracrine fashion. TNF-alpha is a major proinflammatory cytokine, and IL-8 and MCP-1 are major chemoattractants for neutrophils and monocytes/macrophages, respectively. Furthermore, uPA activates matrix metalloproteinase 9 which can truncate and activate IL-8. Thus, the simultaneous induction of TNF-alpha, uPA, IL-8 and MCP-1 may enhance the interaction between tumor and inflammatory/immune cells, and augment cytotoxicity.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Quimiocina CCL2/biossíntese , Doxorrubicina/farmacologia , Interleucina-8/biossíntese , Neoplasias Pulmonares/metabolismo , Fator de Necrose Tumoral alfa/biossíntese , Ativador de Plasminogênio Tipo Uroquinase/biossíntese , Antígenos CD/biossíntese , Northern Blotting , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Citometria de Fluxo , Humanos , Monócitos/metabolismo , Neutrófilos/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Receptores CCR2 , Receptores de Superfície Celular/biossíntese , Receptores de Quimiocinas/biossíntese , Receptores de Interleucina-8A/biossíntese , Receptores do Fator de Necrose Tumoral/biossíntese , Receptores Tipo I de Fatores de Necrose Tumoral , Receptores Tipo II do Fator de Necrose Tumoral , Receptores de Ativador de Plasminogênio Tipo UroquinaseRESUMO
We previously demonstrated doxorubicin-induced urokinase expression in human H69 SCLC cells by the microarray technique using Human Cancer CHIP version 2 (Takara Shuzo, Kyoto, Japan), in which 425 human cancer-related genes were spotted on glass plates (Kiguchi et al., Int J Cancer 2001;93:792-7). Microarray analysis also revealed significant induction of IL-8, a member of the CXC chemokines. We have, therefore, extended the observation by testing the effects of doxorubicin on expression of the chemokine family and provide here definitive evidence that doxorubicin induces IL-8 and MCP-1, one of the CC chemokines, at least in 2 human SCLC cells, H69 and SBC-1. IL-8 antigen levels, measured by ELISA, were markedly increased in both H69 and SBC-1 conditioned media after doxorubicin treatment, in parallel with mRNA levels; and this was dependent on the dose of doxorubicin. The ribonuclease protection assay, using a multiprobe template set for human chemokines, revealed induction of not only IL-8 but also MCP-1 in doxorubicin-treated H69 cells. MCP-1 antigen levels increased approximately 100-fold in doxorubicin-treated H69 cells. RT-PCR using specific primers for MCP-1 suggested that doxorubicin also induced MCP-1 expression in SBC-1 and SBC-3 SCLC cells. Futhermore, CAT analysis using IL-8 promoter implicated the PEA3 transcriptional factor, whose binding site was located immediately upstream of the AP-1 and NF-kappaB binding sites. Thus, it is suggested that doxorubicin induces IL-8 and MCP-1 chemokines in human SCLC cells by activating gene expression, in which at least PEA3 is involved. IL-8 and MCP-1 are major chemoattractants for neutrophils and monocytes/macrophages, respectively; therefore, extensive induction of IL-8 and MCP-1 may provoke the interaction between inflammatory/immune cells and tumor cells under doxorubicin stimulation and influence many aspects of tumor cell biology.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Carcinoma de Células Pequenas/tratamento farmacológico , Quimiocina CCL2/biossíntese , Doxorrubicina/farmacologia , Interleucina-8/biossíntese , Neoplasias Pulmonares/tratamento farmacológico , Northern Blotting , Carcinoma de Células Pequenas/metabolismo , Carcinoma de Células Pequenas/patologia , Quimiocina CCL2/genética , Cloranfenicol O-Acetiltransferase/metabolismo , Primers do DNA/química , Ensaio de Desvio de Mobilidade Eletroforética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Interleucina-8/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , NF-kappa B/genética , NF-kappa B/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Transfecção , Células Tumorais CultivadasRESUMO
All-trans retinoic acid (ATRA) has been shown to induce differentiation of human acute promyelocytic leukaemia (APL) cells and eventual elimination of the malignant clone. Matrix metalloproteinase-9 (MMP-9) is produced by neutrophils and its expression appears to be linked with myeloid cell differentiation. We investigated effects of ATRA on MMP expression in two human myeloid leukaemia cell lines, PL-21 and NB4. Both cells could differentiate into neutrophils after exposure to ATRA. Both the activity and antigen levels of MMP-9 were much higher in NB4 cells than in PL-21 cells. Stimulation with ATRA significantly increased MMP-9 levels approximately three- to fivefold in both PL-21 and NB4-conditioned media. MMP-9 mRNA levels increased in ATRA-treated cells and was almost in parallel with the increase in MMP-9 activity, suggesting that ATRA induced MMP-9 by activating its gene expression. ATRA can induce interleukin 8 (IL-8) in APL cells. IL-8, chemokine for neutrophils and a potent inducer of MMP-9, was also induced by ATRA in PL-21 cells. However, recombinant IL-8 did not induce MMP-9 expression. In addition, a neutralizing antibody against IL-8 did not inhibit ATRA-induced MMP-9 expression in either cell type. These observations suggest that ATRA can induce both MMP-9 and IL-8, but IL-8 is not involved in ATRA-induced MMP-9 expression. As MMP-9 can truncate and activate IL-8, simultaneous induction of MMP-9 and IL-8 by ATRA could activate leucocytes excessively, causing the hyper-inflammatory events in retinoic acid syndrome.