RESUMO
BACKGROUND: The p38 MAPK is constitutively activated in B-NHL cell lines and regulates chemoresistance. Accordingly, we hypothesized that activated p38 MAPK may be associated with the in vivo unresponsiveness to chemotherapy in B-NHL patients. METHODS: Tissue microarrays generated from eighty untreated patients with Diffused Large B Cell Lymphoma (DLBCL) were examined by immunohistochemistry for the expression of p38 and phospho p38 (p-p38) MAPK. In addition, both Bcl-2 and NF-κB expressions were determined. Kaplan Meier analysis was assessed. RESULTS: Tumor tissues expressed p38 MAPK (82 %) and p-p38 MAPK (30 %). Both p38 and p-p38 MAPK expressions correlated with the high score performance status. A significant correlation was found between the expression p-p38 and poor response to CHOP. The five year median follow-up FFS was 81 % for p38(-) and 34 % for p38(+) and for OS was 83 % for p38(-) and 47 % for p38(+). The p-p38(+) tissues expressed Bcl-2 and 90 % of p-p38(-) where Bcl-2(-). The coexpression of p-p38 and Bcl-2 correlated with pool EFS and OS. There was no correlation between the expression of p-p38 and the expression of NF-κB. CONCLUSION: The findings revealed, for the first time, that a subset of patients with DLBCL and whose tumors expressed high p-p38 MAPK responded poorly to CHOP therapy and had poor EFS and OS. The expression of p38, p-p38, Bcl2 and the ABC subtype are significant risk factors both p38 and p-p38 expressions remain independent prognostic factors.
Assuntos
Linfoma Difuso de Grandes Células B/tratamento farmacológico , Linfoma Difuso de Grandes Células B/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Ciclofosfamida/administração & dosagem , Intervalo Livre de Doença , Doxorrubicina/administração & dosagem , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Estimativa de Kaplan-Meier , Linfoma Difuso de Grandes Células B/patologia , Masculino , Pessoa de Meia-Idade , NF-kappa B/genética , Prednisona/administração & dosagem , Prognóstico , Proteínas Proto-Oncogênicas c-bcl-2/genética , Análise Serial de Tecidos , Vincristina/administração & dosagem , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Leishmanioses are chronic parasitic diseases and host responses are associated with pro- or anti-inflammatory cytokines involved, respectively, in the control or exacerbation of infection. The relevance of other inflammatory mediators and stress markers has not been widely studied and there is a need to search for biomarkers to leishmaniasis. In this work, the stress and inflammatory molecules p38 mitogen-activated protein kinase, cyclooxygenase-2, migration inhibitory factor, macrophage inflammatory protein 2, heat shock protein 70 kDa, vascular endothelial factor (VEGF), hypoxia-inducible factors (HIF-1α and HIF-2α), heme oxygenase and galectin-3 expression were assessed immunohistochemically in self-controlled lesions in C57BL/6 mice and severe lesions in Balb/c mice infected with Leishmania amazonensis. The results indicated that the majority of molecules were expressed in the cutaneous lesions of both C57BL/6 and Balb/c mice during various phases of infection, suggesting no obvious correlation between the stress and inflammatory molecule expression and the control/exacerbation of leishmanial lesions. However, the cytokine VEGF was only detected in C57BL/6 footpad lesions and small lesions in Balb/c mice treated with antimonial pentavalent. These findings suggest that VEGF expression could be a predictive factor for murine leishmanial control, a hypothesis that should be tested in human leishmaniosis.
Assuntos
Ciclo-Oxigenase 2/imunologia , Citocinas/imunologia , Inflamação/imunologia , Leishmaniose Cutânea/imunologia , Fator A de Crescimento do Endotélio Vascular/imunologia , Animais , Biomarcadores/análise , Ciclo-Oxigenase 2/biossíntese , Modelos Animais de Doenças , Imuno-Histoquímica , Leishmania/imunologia , Leishmaniose Cutânea/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Óxido Nítrico/biossíntese , Nitroimidazóis/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/imunologiaRESUMO
Increased expression of COX-2 has been linked to inflammation and carcinogenesis. Constitutive expression of COX-2 protects hepatocytes from several pro-apoptotic stimuli. Increased hepatic apoptosis has been observed in experimental models of diabetes. Our present aim was to analyze the role of COX-2 as a regulator of apoptosis in diabetic mouse liver. Mice of C57BL/6 strain wild type (Wt) and transgenic in COX-2 (hCOX-2 Tg) were separated into Control (vehicle) and SID (streptozotocin induced diabetes, 200 mg/kg body weight, i.p.). Seven days post-injection, Wt diabetic animals showed a decrease in PI3K activity and P-Akt levels, an increase of P-JNK, P-p38, pro-apoptotic Bad and Bax, release of cytochrome c and activities of caspases-3 and -9, leading to an increased apoptotic index. This situation was improved in diabetic COX-2 Tg. In addition, SID COX-2 Tg showed increased expression of anti-apoptotic Mcl-1 and XIAP. Pro-apoptotic state in the liver of diabetic animals was improved by over-expression of COX-2. We also analyzed the roles of high glucose-induced apoptosis and hCOX-2 in vitro. Non-transfected and hCOX-2-transfected cells were cultured at 5 and 25 mM of glucose by 72 h. At 25 mM there was an increase in apoptosis in non-transfected cells versus those exposed to 5 mM. This increase was partly prevented in transfected cells at 25 mM. Moreover, the protective effect observed in hCOX-2-transfected cells was suppressed by addition of DFU (COX-2 selective inhibitor), and mimicked by addition of PGE(2) in non-transfected cells. Taken together, these results demonstrate that hyperglycemia-induced hepatic apoptosis is protected by hCOX-2 expression.
Assuntos
Apoptose , Ciclo-Oxigenase 2/metabolismo , Hiperglicemia/metabolismo , Fígado/metabolismo , Animais , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular , Ciclo-Oxigenase 2/genética , Citocromos c/biossíntese , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Fígado/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Estreptozocina , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/biossíntese , Proteína X Associada a bcl-2/biossíntese , Proteína de Morte Celular Associada a bcl/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/biossínteseRESUMO
We previously found that prenatal hypoxia induces a significant increase in the levels of active Caspase 3 at 60 min post-hypoxia (p-h) and in the number of TUNEL-positive pyknotic cells, which peaks at 6h p-h. The aim of this work was to study alterations in MAPKs pathways and the effect of specific inhibitors of the JNK (SP600125) and p38 (SB203580) pathways following acute hypoxia in chick optic lobe at embryonic day (ED) 12. To this end, JNK, p38 and ERK1-2 protein kinase expression levels were determined by Western blot in both their active and inactive forms, evaluated at successive p-h times. At 10 and 30 min p-h the P-JNK/JNK ratio was 1.912+/-0.341 and 1.920+/-0.304, respectively. Concomitantly, at 0 min p-h the P-p38/p38 ratio was 1.657+/-0.203. Lastly, the P-ERK/ERK ratio proving non-significant throughout. When inhibitors for JNK and p38 were used, we observed a decrease in the values of active Caspase 3 at 60 min p-h, which correlated with the control values in the parameters of TUNEL-positive cells at 6h p-h. Analysis for P-ATF-2 demonstrated an increase in hypoxic embryos compared to control ones which was reverted in a dose-dependent manner with the use of both inhibitors. All these results indicate that at ED 12, acute hypoxia might be differentially activating JNK and p38 pathways, without affecting the ERK pathway, which in turn would be activating Caspase 3, thus leading to cell death by apoptosis. Furthermore, JNK and p38 activation precede in time the programmed cell death induced by hypoxia.
Assuntos
Apoptose/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/biossíntese , Hipóxia Fetal/enzimologia , Hipóxia Fetal/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Animais , Apoptose/genética , Western Blotting , Caspase 3/biossíntese , Caspase 3/genética , Embrião de Galinha , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Hipóxia Fetal/genética , Marcação In Situ das Extremidades Cortadas , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
The octapeptide angiotensin II (ANG II) can modulate cardiac contractility and is increased in heart failure, where contractile function is impaired. In rat cardiac myocytes, 1 microM of ANG II produces a negative inotropic effect (NIE) (24.6 +/- 5% reduction). However, the subcellular signaling involved in this effect remains elusive. We examined the mechanisms and signaling events involved in the reduction in contractile function induced by the peptide in indo-1-loaded rat cardiomyocytes. The results showed that the NIE of ANG II was not associated with a parallel decrease in the intracellular Ca2+ transient, indicating that a decrease in myofilament responsiveness to Ca2+ underlies the reduction in contractility. We assessed the role of PKC, tyrosine kinases, reactive oxygen species (ROS), and mitogen-activated protein kinases (MAPKs) in the NIE of the peptide. Pretreatment of cells with the NAD(P)H oxidase inhibitor diphenyleneiodonium chloride or with the superoxide scavenger 4,5-dihydroxy-1,3-benzene-disulfonic acid did not affect the ANG II-induced NIE. Moreover, ANG II-induced ROS production, after 20 min of incubation with the peptide, could not be detected with the use of either the fluorophore 5-(6)-chloromethyl-2',7'-dichlorodihydrofluorecein diacetate or lucigenin-enhanced chemiluminescence. In contrast, the ANG II-induced NIE was abrogated by the inhibitors of PKC (calphostin C), tyrosine kinase (genistein), and p38 MAPK (SB-202190). Furthermore, the NIE was significantly exacerbated (60 +/- 10% reduction) by p38 MAPK overexpression. These results exclude the participation of ROS in the NIE of the peptide and point to PKC and tyrosine kinase as upstream mediators. Furthermore, they reveal p38 MAPK as the putative effector of the reduction in myofilament responsiveness to Ca2+ and the decrease in contractility induced by the peptide.
Assuntos
Angiotensina II/farmacologia , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologia , Animais , Cálcio/administração & dosagem , Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Depressão Química , Genisteína/farmacologia , Ventrículos do Coração/citologia , Imidazóis/farmacologia , Naftalenos/farmacologia , Proteína Quinase C/fisiologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Piridinas/farmacologia , Ratos , Ratos Wistar , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/biossínteseRESUMO
Lead (Pb2+) is a neurotoxic trace metal, widespread in aquatic environment that can change physiologic, biochemical and behavioral parameters in diverse fish species. Chemical exposure may drive modulation of mitogen-activated protein kinases (MAPKs) that are a family of highly conserved enzymes which comprise ubiquitous groups of signaling proteins playing critical regulatory roles in cell physiology. Extracellular signal-regulated kinases (ERK1/2) and p38(MAPK) control complex programs such as gene expression, embryogenesis, cell differentiation, cell proliferation, cell death and synaptic plasticity. Little information is available about MAPKs in aquatic organisms and their modulation by trace metals. The aim of this work was to determine the modulation of ERK1/2 and p38(MAPK) phosphorylation by Pb2+ in vivo and in vitro, in cerebellar slices of the catfish, Rhamdia quelen. In the in vitro model, slices were incubated for 3 h with lead acetate (1-10 microM). In the in vivo studies, the animals were exposed for 2 days to lead acetate (1 mg L(-1)). ERK1/2 and p38(MAPK) (total and phosphorylated forms) were immunodetected in cerebellar slices by Western blotting. Pb2+ added in vitro at 5 and 10 microM increased significantly the phosphorylation of both MAPKs. The in vivo exposed animals also showed a significant increase of ERK1/2 and p38(MAPK) phosphorylation without changes in the total content of the enzymes. In conclusion, the present work indicates that it is possible to evaluate the ERK1/2 and p38(MAPK) activation in the central nervous system (CNS) of a freshwater fish largely distributed in South America. Moreover, Pb2+, an important environmental pollutant may activate in vitro and in vivo ERK1/2 and p38(MAPK) enzymes. These findings are important considering the functional and ecologic implications associated to Pb2+ exposure of a freshwater fish species, such as R. quelen, and the roles of ERK1/2 and p38(MAPK) in the control of brain development, neuroplasticity and cell death.
Assuntos
Peixes-Gato/metabolismo , Cerebelo/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Compostos Organometálicos/toxicidade , Poluentes Químicos da Água/toxicidade , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Animais , Western Blotting/veterinária , Cerebelo/enzimologia , Exposição Ambiental , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Compostos Organometálicos/administração & dosagem , Proteínas Quinases p38 Ativadas por Mitógeno/biossínteseRESUMO
Previous studies have emphasized the role of Toll-like receptors (TLRs) and myeloid differentiation factor 88 (MyD88) during infection with protozoan parasites. TLR2 was shown to be important for induction of cytokine synthesis by macrophages exposed to the purified glycosylphosphatidylinositol (GPI)-anchored mucin-like glycoproteins of Trypanosoma cruzi trypomastigotes (tGPIm). On the other hand, MyD88(-/-) mice, but not TLR2(-/-) mice, showed impaired cytokine production and resistance to infection with T. cruzi parasites. Here we evaluate the importance of MyD88 and TLR2 in MAPK activation and cytokine synthesis by macrophages exposed to live T. cruzi parasites and compared to tGPIm. The absence of MAPK phosphorylation in TLR2- and MyD88-deficient macrophages exposed to tGPIm correlated with the incapacity to induce cytokine release in these cells. In contrast, activation of MAPK and synthesis of pro-inflammatory cytokines were not abrogated in TLR2-deficient macrophages exposed to live T. cruzi parasites. We also showed that pretreatment with tGPIm significantly reduces cytokine release by macrophages in response to T. cruzi in a TLR2-dependent manner. Consistently, TLR2(-/-) mice were shown to produce enhanced levels of cytokines upon in vivo challenge with T. cruzi parasites. Together, these results suggest the involvement of additional TLR(s) in the pro-inflammatory response of macrophages to whole parasites, and that, in vivo, TLR2 may have a predominant immunoregulatory role during acute infection with T. cruzi parasites.