Nuclear Factor-κB Signaling Mediates Antimony-induced Astrocyte Activation / 生物医学与环境科学（英文）

Objective@#Antimony (Sb) has recently been identified as a novel nerve poison, although the cellular and molecular mechanisms underlying its neurotoxicity remain unclear. This study aimed to assess the effects of the nuclear factor kappa B (NF-κB) signaling pathway on antimony-induced astrocyte activation.@*Methods@#Protein expression levels were detected by Western blotting. Immunofluorescence, cytoplasmic and nuclear fractions separation were used to assess the distribution of p65. The expression of protein in brain tissue sections was detected by immunohistochemistry. The levels of mRNAs were detected by Quantitative real-time polymerase chain reaction (qRT-PCR) and reverse transcription-polymerase chain reaction (RT-PCR).@*Results@#Antimony exposure triggered astrocyte proliferation and increased the expression of two critical protein markers of reactive astrogliosis, inducible nitric oxide synthase (iNOS) and glial fibrillary acidic protein (GFAP), indicating that antimony induced astrocyte activation @*Conclusion@#Antimony activated astrocytes by activating the NF-κB signaling pathway.

Transforming growth factor-β-activated kinase 1 and pathological myocardial hypertrophy / 生理学报

The transforming growth factor-β-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family. TAK1 plays important roles in many biological functions. Cardiac hypertrophy can be identified as physiological or pathological myocardial hypertrophy. TAK1 not only participates in the development of normal myocardium, but also plays an important role in regulating the occurrence and development of pathological myocardial hypertrophy. Angiotensin II (Ang II) or pressure overload induces pathological cardiac hypertrophy through different ways, such as hypoxia-inducible factor-1α (HIF-1α)-mediated transcriptional expression of TAK1, or transforming growth factor-β1 (TGF-β1)-, thyroid hormone-, ubiquitin protease-mediated TAK1 phosphorylation or ubiquitination. This article reviews the role of TAK1 in the occurrence and development of pathological myocardial hypertrophy and discusses the potential of TAK1 as an important target for the prevention and treatment of clinical myocardial hypertrophy.

Modificações pós-traducionais durante o processo de amastigogênese do Trypanosoma cruzi / Post-translational modifications during the amastigogenesis of Trypanosoma cruzi

A doença de Chagas é uma doença negligenciada causada pelo protozoário Trypanosoma cruzi constituindo-se em um problema de saúde pública em vários países da América Latina. No seu complexo ciclo de vida, o protozoário passa por quatro estágios diferentes: tripomastigota metacíclica, amastigota, tripomastigota sanguíneo e epimastigota, que permitem sua sobrevivência nos diferentes ambientes com os quais o parasita entra em contato. A diferenciação dos tripomastigotas de T. cruzi em amastigotas (amastigogênese) ocorre com grandes mudanças morfológicas, estruturais e metabólicas no parasita e pode ser reproduzido in vitro por exemplo, pela acidificação do meio extracelular. Apesar dos vários trabalhos descritos na literatura, o processo ainda não é totalmente compreendido. A participação de NO na transdução de sinal durante a amastigogênese, sugerida por dados não publicados de nosso grupo, assim como a via de sinalização dependente de AMPc, foram o foco do presente estudo. A indução da amastigogênese foi obtida por incubação de tripomastigotas em meio de cultura acidificado (pH 6,0) e os parâmetros estudados comparados com parasitas controle (meio de cultura, pH 7,4). Estudamos a variação no perfil de nucleotídios cíclicos (AMPc, GMPc), de quinases (PKA, MAPK- ERK1/2), de uma fosfatase (PP2A), assim como o perfil de proteínas fosforiladas, S-nitrosiladas e nitradas até 6 h do início da amastigogênese. O processo foi dividido nas etapas: inicial (até 60 minutos) e tardio (em torno de 3-4 h), caracterizados por um aumento de formas amastigotas na etapa tardia. Houve um aumento de aproximadamente 17 vezes no nível de AMPc nos primeiros 15 minutos da amastigogênese (meio pH 6,0), seguido por aumento discreto no nível de PKA fosforilada, utilizado como indicador de atividade enzimática, este mais evidente na etapa tardia (360 minutos). Quanto à subunidade catalítica fosforilada da MAPK (ativa), há uma aparente diminuição no nível de fosforilação na fase inicial (30 minutos) e aumento na etapa tardia (120 minutos) do processo de amastigogênese. Quanto ao perfil geral de fosforilação de proteínas, há uma diminuição de fosforilação em torno de 30 minutos, seguida de aumento de fosforilação em proteínas de aproximadamente 5 e 100 kDa, mas de maneira geral, não se observaram grandes mudanças nesse perfil com a metodologia utilizada. Quanto às modificações por NO e seus derivados, foram observadas modificações por S-nitrosilação e nitração das proteínas, além do aumento de GMPc em torno de 60 minutos. Embora essas modificações modulem a atividade biológica de uma grande diversidade de proteínas, seu papel biológico não foi explorado.8 Em resumo, nossos resultados apontam para uma variação no perfil de fosforilação, S-nitrosilação e nitração de proteínas, além do aumento de AMPc e GMPc ao longo do processo de amastigogênese in vitro, com a via de sinalização dependente de quinases/ fosfatases e de óxido nítrico ocorrendo ao longo do processo de amastigogênese

Chagas disease is a neglected disease caused by the parasite Trypanosoma cruzi and is a public health problem in several Latin American countries. In its complex life cycle, the protozoan goes through four different stages: metacyclic trypomastigote, amastigote, blood trypomastigote and epimastigote, which allow its survival in the different environments which the parasite comes into contact. The differentiation of T. cruzi trypomastigotes into amastigotes (amastigogenesis) occurs with large morphological, structural and metabolic changes in the parasite and can be reproduced in vitro by, for example, acidification of the extracellular medium. Despite the many data described in the literature, the process is not yet fully understood. The participation of NO in signal transduction during amastigogenesis, suggested by unpublished data from our group, as well as the cAMP-dependent signaling pathway, were the focus of the present study. The induction of amastigogenesis was obtained by incubating trypomastigotes in acidified culture medium (pH 6.0) and the studied parameters compared with control parasites (culture medium, pH 7.4). We studied the variation in the profile of cyclic nucleotides (cAMP, cGMP), kinases (PKA, MAPK-ERK1 / 2), phosphatase (PP2A), as well as the profile of phosphorylated, S-nitrosylated and nitrated proteins up to 6 h. onset of amastigogenesis. The process was divided into early (up to 60 minutes) and late (around 3-4 hours), characterized by an increase in amastigote forms in the late stage. There was an approximately 17-fold increase in cAMP level in the first 15 minutes of amastigogenesis (pH 6.0 medium), followed by a slight increase in phosphorylated PKA level, most evident in the late stage (360 minutes). As for the phosphorylated catalytic subunit of MAPK (active), there is an apparent decrease in the phosphorylation level in the early phase (30 minutes) and increase in the late stage (120 minutes) of the amastigogenesis process. As for the general protein phosphorylation profile, there is a decrease in phosphorylation around 30 minutes, followed by an increase in phosphorylation of proteins (approximately 5 and 100 kDa), but overall, no major changes were observed in this profile with the methodology used. As for modifications by NO and its derivatives, modifications were observed by S-nitrosylation and protein nitration, besides the increase of cGMP around 60 minutes. Although these modifications modulate the biological activity of a wide range of proteins, their biological role has not been explored. In summary, our results point to a variation in phosphorylation, S-nitrosylation and nitration profile of proteins, as well as an increase in cAMP and cGMP along the amastigogenesis process, implicating kinases / phosphatases and nitric oxide dependent signaling pathways in this differentiation

Avaliação do potencial de superação da quimioresistência do melanoma aos inibidores de BRAFV600E (Vemurafenibe) e de MEK (Trametinibe) utilizando terapia combinatória com 4-nerolidilcatecol (4-NC) / Evaluation of the potential of overcoming the chemoresistance of melanoma to BRAFV600E (Vemurafenib) and MEK (Trametinib) inhibitors using combinatory therapy with 4-nerolidylcatechol (4-NC)

Embora o melanoma represente apenas 4% das neoplasias malignas da pele, é considerado a mais grave por ser altamente etal. Em virtude da via MAPK (Mitogen activated protein kinase) estar intimamente ligada ao descontrole da proliferação celular, especialmente em melanoma, esta via se tornou um alvo para o desenvolvimento de terapias direcionadas a oncogenes, como os potentes quimioterápicos Vemurafenibe (inibidor da mutação V600E em BRAF - BRAFV600E) e Trametinibe (inibidor de MEK). Cada vez mais, melhores taxas de respostas vêm sendo alcançadas com os novos medicamentos, porém a maioria dos pacientes está sujeita a recidivas após 7 meses de tratamento devido ao desenvolvimento de quimioresistência, justificando a constante busca por novos compostos terapêuticos. Dados de nosso laboratório indicam que 4-nerolidilcatecol (4-NC) induz aumento na expressão de p53, produção de ROS e dano ao DNA, culminando em apoptose dependente de caspase-3 em células de melanoma por ser um inibidor proteassomal. Além disto, o 4-nerolidilcatecol (4- NC) demonstrou efeito inibitório na proliferação de células de melanoma em modelo de cultura organotípica de pele. Desta forma, este projeto visa avaliar a possibilidade de superação da quimioresistência aos inibidores de BRAF e de MEK, utilizando terapias combinatórias com 4-NC em células de melanoma humano resistentes a estes inibidores. Primeiramente, linhagens celulares de melanoma resistentes aos inibidores de BRAF (R) e BRAF/MEK (DR) foram geradas a partir de células parentais BRAF mutadas (P) e caracterizadas por MTT, microscopia de fluorescência e western blotting. Estas células foram submetidas ao tratamento com 4-NC que apresentou citotoxicidade na concentração de 30µM, inibição de formação de colônias e diminuição na invasão em modelos in vitro de culturas 2D e 3D em todas as linhagens estudadas (P, R e DR). O 4-NC foi ainda capaz de induzir estresse de retículo endoplasmático com indução de apoptose. Visando a explorar o efeito terapêutico in vivo do 4-NC, outro estudo foi conduzido em animais submetidos a enxerto xenográfico com células parentais de melanoma NRAS mutadas. Após desenvolvimento do tumor, os animais foram tratados 3 vezes por semana durante 3 semanas com 4-NC (10 mg/kg) por via i.p. O 4-NC foi capaz de inibir em até 4 vezes o crescimento dos tumores xenográficos (4/10) quando comparado com os controles, com remissão completa do tumor em um animal. A expressão de p53 e PARP clivada foi aumentada nos tumores dos animais tratados, sugerindo apoptose. A expressão gênica de MMP2 e MMP14 estava diminuída nas mesmas amostras, demonstrando o papel do 4-NC na inibição da invasão do melanoma in vivo. Finalmente, a toxicidade sistêmica do 4-NC foi avaliada nas mesmas doses empregadas no ensaio in vivo de tumorigênese. A baixa toxicidade observada nos ensaios toxicológicos com tratamentos sub-crônicos com 4-NC e a citotoxicidade demonstrada em modelos xenográficos nos leva a considerar este composto como promissor para estudos futuros e sua aplicação no tratamento do melanoma cutâneo humano, incluindo pacientes resistentes aos inibidores de BRAF e MEK

Melanoma accounts for only 4% of malignant neoplasms of the skin, but is considered the most serious because it is highly deadly. Because the MAPK (Mitogen activated protein kinase) pathway is closely linked to the lack of control of cell proliferation, especially in melanoma, this pathway has become a target for the development of oncogene-targeted therapies, such as the potent chemotherapeutic agents Vemurafenib (V600E mutation inhibitor in BRAF - BRAFV600E) and Trametinib (MEK inhibitor). Increasingly, better response rates have been achieved with the new drugs, but most patients are subject to relapses after 7 months of treatment due to several mechanisms, which justify the constant search for new therapeutic compounds. Data from our laboratory indicate that 4-nerolidylcatechol (4-NC) induces increased p53 expression, ROS production and DNA damage, culminating in caspase-3 dependent apoptosis in melanoma cells. The 4-NC compound demonstrated an inhibitory effect on melanoma cell proliferation in an organotypic skin culture model. Thus, this project aims to evaluate the possibility of overcoming the existing chemoresistance to BRAF and MEK inhibitors, using 4-NC combinatory therapies in human melanoma cells resistant to these inhibitors. Firstly, melanoma cell lines resistant to BRAF (R) and BRAF / MEK (DR) inhibitors were generated from naive cells mutated BRAF (N) and characterized by MTT, fluorescence microscopy and western blotting. These cells were submitted to 4-NC treatment that showed cytotoxicity with 30 µM, inhibition of colony formation and decrease in invasion in 2D and 3D in vitro models in all cell line studied (N, R and DR). Furthermore, 4-NC was able to induce endoplasmic reticulum stress with apoptosis induction. In order to explore the in vivo therapeutic effect of 4-NC, an additional study was conducted using xenograft model with NRAS-mutated melanoma cell line. After tumor development, the animals were treated 3 times per week for 3 weeks with 4-NC (10 mg / kg) by i.p. injection. 4-NC was able to inhibit up to 4- fold the growth of xenograft tumors (4/10) when compared to controls, with complete tumor remission in one animal. Cleaved PARP and p53 expression were increased in the tumors of treated animals, suggesting apoptosis. MMP2 and MMP14 gene expression were decreased in the same samples, demonstrating the role of 4-NC in inhibiting melanoma invasion in vivo. Finally, the systemic toxicity of 4-NC was evaluated at the same doses employed in the in vivo tumorigenesis assay. The low toxicity observed in the toxicological assays with sub-chronic 4- NC treatments and the demonstrated cytotoxicity in xenograft models leads us to consider this compound as promising for future studies and its application in the treatment of cutaneous human melanoma, including patients resistants to BRAF and MEK inhibitors

Role of MAPK Signaling Pathways in Regulating the Hydrophobin Cryparin in the Chestnut Blight Fungus Cryphonectria parasitica

We assessed the regulation of cryparin, a class II hydrophobin, using three representative mitogen-activated protein kinase (MAPK) pathways in Cryphonectria parasitica. Mutation of the CpSlt2 gene, an ortholog of yeast SLT2 in the cell wall integrity (CWI) pathway, resulted in a dramatic decrease in cryparin production. Similarly, a mutant of the CpBck1 gene, a MAP kinase kinase kinase gene in the CWI pathway, showed decreased cryparin production. Additionally, mutation of the cpmk1 gene, an ortholog of yeast HOG1, showed decreased cryparin production. However, mutation of the cpmk2 gene, an ortholog of yeast Kss1/Fus3, showed increased cryparin production. The easy-wet phenotype and accumulation of the cryparin transcript in corresponding mutants were consistent with the cryparin production results. In silico analysis of the promoter region of the cryparin gene revealed the presence of binding motifs related to downstream transcription factors of CWI, HOG1, and pheromone responsive pathways including MADS-box- and Ste12-binding domains. Real-time reverse transcriptase PCR analyses indicated that both CpRlm1, an ortholog of yeast RLM1 in the CWI pathway, and cpst12, an ortholog of yeast STE12 in the mating pathway, showed significantly reduced transcription levels in the mutant strains showing lower cryparin production in C. prasitica. However, the transcription of CpMcm1, an ortholog of yeast MCM1, did not correlate with that of the mutant strains showing downregulation of cryparin. These results indicate that three representative MAPK pathways played a role in regulating cryparin production. However, regulation varied depending on the MAPK pathways: the CWI and HOG1 pathways were stimulatory, whereas the pheromone-responsive MAPK was repressive.

Caracterização do perfil de resistência da linhagem de leucemia mieloide crônica K-IM, resistente ao imatinibe / [Characterization of the resistance profile of the K-IM chronic myeloid leukemia strain, resistant to imatinib]

O advento da terapia alvo-específica com o imatinibe (IM) transformou o panorama da resposta ao tratamento, progressão e sobrevida dos pacientes de leucemia mieloide crônica (LMC). Apesar do sucesso e do desenvolvimento de inibidores de tirosina-quinase (TKIs) mais potentes, persiste a problemática da resistência ao tratamento. A relevância do estudo dos mecanismos de resistência aos TKIs atualmente reside na frequência das falhas terapêuticas não relacionadas à mutação em BCR-ABL, o principal alvo da terapia. Tendo por finalidade estudar os diversos mecanismos que cooperam para a aquisição de resistência, foi desenvolvida em nosso laboratório uma linhagem de LMC resistente ao IM. A linhagem resistente, denominada K-IM, foi selecionada pelo cultivo dalinhagem K562 em concentrações crescentes de IM, alcançando 1,0 μM do fármaco. Sem apresentar mutação no domínio quinase de BCR-ABL, ela se constitui um bom modelo para oestudo dos outros mecanismos de resistência ao IM. A linhagem apresentou aumento nos níveis deRNA mensageiro (RNAm) de BCR-ABL que não se traduziu em aumento da atividade de Bcr-Abl ou no impedimento da sua inibição pelo tratamento com IM. A linhagem K-IM se mostrou significativamente mais resistente do que a parental. Embora o tratamento com 1,0 μM de IM tenha promovido um acúmulo de células nas fases G0/G1 do ciclo celular, não foi acompanhado deindução à morte celular nem impediu o aumento do número de células em cultura. Foram avaliadas as proteínas transportadoras de efluxo por serem determinantes para a resistência à múltiplas drogas, porém a sua atividade não foi observada na linhagem K-IM...

The advent of target-specific therapy with imatinib (IM) has transformed the scenario of response to treatment, progression and survival of patients with chronic myeloid leukemia (CML). Despite the success and development of more potent tyrosine kinase inhibitors (TKIs), the problem of resistance to treatment remains. The relevance of the study of mechanisms of resistance to TKIs currently lies in the frequency of therapeutic failures unrelated to mutation in BCR-ABL, the main target of therapy. Aiming to study the various mechanisms that cooperate for the acquisition of resistance, a CML cell line resistant to IM was developed in our laboratory. The resistant cell line, called K-IM, was selected by culturing the K562 cell line at increasing concentrations of IM, reaching 1.0 μM of the drug. Without presenting mutation in the BCRABL kinase domain, it constitutes a good model for the study of the other mechanisms ofresistance to IM. The cell line showed an increase in BCR-ABL messenger RNA (mRNA) levels which did not result in increased Bcr-Abl activity or in the impairment of its inhibition by IMtreatment. The K-IM cell line was significantly more resistant to IM than the parental cell line. Although treatment with 1.0 μM of IM promoted cell accumulation in the G0/G1 phases of the cell cycle, it was not accompanied by induction of cell death nor prevented the increase in the number of cells in culture. The efflux transporter proteins were evaluated because they aredeterminant for multidrug resistance, but their activity was not observed in the K-IM cell line. Since the antiapoptotic proteins XIAP and survivin are studied as chemoresistance factors, their mRNA and protein levels were evaluated. K-IM showed similar levels to K562 of XIAP mRNA,but showed higher survivin levels in both instances, suggesting that this protein plays a role in its resistance...

Mechanism of colon cancer cell apoptosis induced by telocinobufagin: role of oxidative stress and apoptosis pathway / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effects of telocinobufagin on viability and apoptosis of colorectal cancer (CRC) cells and explore the mechanism of telocinobufagin-induced apoptosis.</p><p><b>METHODS</b>MTT assay was performed to detect the viability of CRC cells exposed to telocinobufagin. Nuclear staining with Hoechst 33342 and flow cytometry were used to analyze the cell death of CRC cells. Expressions of proteins related with cell apoptosis and oxidative stress were determined with Western blotting.</p><p><b>RESULTS</b>Telocinobufagin decreased the viability of CRC cells in a time- and dose-dependent manner. The presence of karyopycnosis and apoptotic bodies together with the results of flow cytometry suggested that telocinobufagin induced cell apoptosis to cause cell death. Western blotting showed that telocinobufagin exposure of the cells resulted in upregulated p53 and Bax protein expressions and promoted cleavage of caspase 9 and PARP. Telocinobufagin induced phosphorylation of Bad and PARP cleavage, and suppressed phosphorylation of IKBα and TAK1 and expression of survivin in the cells.</p><p><b>CONCLUSION</b>Telocinobufagin can decrease the viability of CRC cells by inducing cell apoptosis, which involves p53-mediated Bax activation and inhibition of the IAP pathway.</p>

Cellular model of neuronal atrophy induced by DYNC1I1 deficiency reveals protective roles of RAS-RAF-MEK signaling

Neuronal atrophy is a common pathological feature occurred in aging and neurodegenerative diseases. A variety of abnormalities including motor protein malfunction and mitochondrial dysfunction contribute to the loss of neuronal architecture; however, less is known about the intracellular signaling pathways that can protect against or delay this pathogenic process. Here, we show that the DYNC1I1 deficiency, a neuron-specific dynein intermediate chain, causes neuronal atrophy in primary hippocampal neurons. With this cellular model, we are able to find that activation of RAS-RAF-MEK signaling protects against neuronal atrophy induced by DYNC1I1 deficiency, which relies on MEK-dependent autophagy in neuron. Moreover, we further reveal that BRAF also protects against neuronal atrophy induced by mitochondrial impairment. These findings demonstrate protective roles of the RAS-RAF-MEK axis against neuronal atrophy, and imply a new therapeutic target for clinical intervention.

Morphology of Hypertrophic Scar Tissues and Expressions of Vascular Endothelial Growth Factor and Transforming Growth Factor Beta Activated Kinase 1 in These Tissues / 中国医学科学院学报

<p><b>OBJECTIVE</b>To observe the morphology of hypertrophic scar tissue and explore the expressions and distribution of vascular endothelial growth factor (VEGF) and transforming growth factor beta activated kinase 1(TAK1 )in these tissues.</p><p><b>METHOD</b>Hematoxylin-eosin staining, Masson staining,immunofluorescence,and real-time polymerase chain reaction were used to detect the localization and expression of VEGF and TAK1 in 15 hypertrophic scar tissues and 10 normal skin tissues.</p><p><b>RESULTS</b>Morphological observation showed that the dermal fibroblasts in hypertrophic scar were disorderly and densely arranged (compared to the normal skin). Immunofluorescence displayed that the expressions of VEGF and TAK1 in hypertrophic scar tissue were higher than in normal skin tissues. Real-time polymerase chain reaction showed the mRNA expressions of both VEGF and TAK1 were significantly higher in hypertrophic scar tissue than in normal tissue (P<0.01, P<0.05,respectively).</p><p><b>CONCLUSIONS</b>Hypertrophic scar tissue has higher collagen fibrosis degree and higher TAK1 and VEGF expressions than the normal skin. VEGF and TAK1 can be used as the reference indicators for the diagnosis and differential diagnosis of hypertrophic scar and serve as new therapeutic targets.</p>

Expression of TRAF-6,TAK1 and TGF-β mRNA in peripheral blood mononuclear cells of patients with diffuse large B cell lymphoma before and after chemotherapy / 中国实验血液学杂志

This study was purposed to detect the expression levels of TRAF6, TAK1 and TGF-β mRNA in peripheral blood mononuclear cell (PBMNC) of patients with diffuse large B cell lymphoma (DLBCL) before and after chemotherapy, and to explore the effect of chemotherapy on the activity of TRAF6/TAK1 signal pathway. The expression levels of TRAF-6, TAK1 and TGF-β mRNA in PBMNC of 38 patients with DLBCL were detected by using the quantitative real time PCR before treatment or after two cycles of chemotherapy, 12 healthy people were served as the control. The results showed that the expression levels of TRAF-6, TAK1 and TGF-β mRNA in PBMNC of DLBCL patients' were higher than those in healthy people. Before treatment, the expression levels of TRAF-6 and TAK1 mRNA had no significant difference as compared with healthy people (P > 0.05); after chemotherapy, the expression levels of these two genes significantly increased, and the differences both had statistically significant as compared with healthy people (P < 0.05); meanwhile the increased expression levels of these two genes after chemotherapy had statistically significant difference as compared with levels before treatment (P < 0.05) , and those expression levels were positively correlated. While the expression level of TGF-β mRNA decreased after chemotherapy as compared with level before treatment, and the differences had statistically significantse(P < 0.05). It is concluded that the activity of TRF6/TAK1 signal pathways in PBMNC of DLBCL patients' significantly increases after chemotherapy, while the expression level of TGF-β mRNA after chemotherapy is abviously lower than level before treatment.

Inhibitory effect of PD98059 on MAPK signaling pathway in acute lymphocytic leukemia cells / 中国实验血液学杂志

This study was purposed to investigate the effect of blocking Ras/Erk signaling pathway on expression of important transcription factor c-fos, c-jun and TAK1 gene in primary acute lymphocytic leukemia (ALL) cells. The best effective concentration and effect time of PD98059 were screened; the expression levels of c-fos, c-jun and TAK1 in primary cultured cells of normal persons, primary cultured ALL cells and primary cultured ALL cells treated by PD98059 were detected by SYBR GreenI real-time quantitative-PCR. The results showed that before treatment by PD98059 the expression levels of c-fos and TAK1 mRNA were significantly up-regulated in primary cultured ALL cells as compared with primary cultured cells of normal persons (P = 0.014 and P = 0.017 respectively). After treatment by PD98059, the expression levels of c-fos, c-jun mRNA decreased in all 7 serum samples, while expression of TAK1 was down-regulated in 5 samples, and up-regulated in 2 samples. After treatment with PD98059, there was no statistical difference of c-fos, c-jun and TAK1 expression levels in primary cultured ALL cells and primary cultured normal cells. It is concluded that the c-fos and TAK1 activity of primary cultured ALL cells increases, and blocking the Ras/Erk signaling pathway of ALL cells can lead to obvious decrease of important transcription factors c-fos, c-jun, TAK1 genes expression.

Mechanism about therapeutic effect of meisoindigo on psoriasis via down-regulation of the TLR4-TAK-NF-kappaB pathways / 药学学报

Meisoindigo is an indigo natural derivative commonly used in anti-cancer therapy. In the clinical application, it was also found to have good therapeutic effect on psoriasis. In order to further understand its mechanism of action, human normal keratinocyte cell line HaCaT and RAW 264.7 were used to identify if meisoindigo could affect the inflammatory factors such as NO and other important cytokines which were highly involved in psoriasis. Our results indicated that meisoindigo decreased the production of NO in LPS-stimulated RAW 264.7 cells and reduced the expression of cytokines in LPS-stimulated HaCaT cells. And TLR4-TAK-NF-kappaB was a possible pathway mainly involved in the attenuation of iNOS and pro-inflammatory cytokine production by meisoindigo, which may take part in the therapeutic effect of meisoindigo on psoriasis.

Effect of TAK1 gene silencing on the apoptosis of Kasumi-1 cells induced by arsenic trioxide / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of transforming growth factor-β activated kinase-1 (TAK1) gene silencing on the proliferation and apoptosis of Kasumi-1 cells induced by arsenic trioxide (As₂O₃).</p><p><b>METHODS</b>Acute myeloid leukemia with t(8;21) cell line Kasumi-1 cells were treated with As₂O₃ or in combination with TAK1 siRNA interference technology. The experiment was divided into four groups: Kasumi-1 cells without any treatment, TAK1 specific siRNA transfection alone, Kasumi-1 cells treated with different concentration of As₂O₃, TAK1siRNA transfection combined with As₂O₃. CCK-8 was used to detect the cell viability. The expression of phosphorylated c-Jun N-terminal kinase (P-JNK) was determined by Western Blot. Cell apoptosis and growth were examined by morphological and colony formation assay.</p><p><b>RESULTS</b>After Kasumi-1 cells were treated with As₂O₃, the rate of cell inhibition was concentration-dependent, and the 50% inhibitory concentration was 3.5 μmol/L. The highest expression level of P-JNK appeared in 30 minutes after cells were treated with As₂O₃. The apoptosis rates of Kasumi-1 cells without any treatment, TAK1 siRNA interference alone group, As₂O₃ alone group and the combined group were (5.02 ± 1.13)%, (6.18 ± 0.28)%, (48.33 ± 2.70)% and (86.07 ± 2.21)%; colony formation rates were (73.83 ± 2.78)%, (76.03 ± 1.46)%, (55.07 ± 1.50)% and (22.20 ± 1.15)%; apoptosis rate of TAK1 siRNA group and the untreated group has no significant difference (P = 0.052); colony formation rate between TAk1 siRNA group and the untreated group has no significant difference (P = 0.179), but the difference in other groups was significant (P = 0.000).</p><p><b>CONCLUSION</b>Silencing the expression of TAK1 can enhance the anti-proliferative and pro-apoptotic effect of As₂O₃ on Kasumi-1 cells, and its mechanism may be through the TAK1 downstream JNK signal pathway.</p>

TGF-beta-activated kinase-1: New insights into the mechanism of TGF-beta signaling and kidney disease

Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that regulates a wide variety of cellular functions, including cell growth, cellular differentiation, apoptosis, and wound healing. TGF-beta1, the prototype member of the TGF-beta superfamily, is well established as a central mediator of renal fibrosis. In chronic kidney disease, dysregulation of expression and activation of TGF-beta1 results in the relentless synthesis and accumulation of extracellular matrix proteins that lead to the development of glomerulosclerosis and tubulointerstitial fibrosis, and ultimately to end-stage renal disease. Therefore, specific targeting of the TGF-beta signaling pathway is seemingly an attractive molecular therapeutic strategy in chronic kidney disease. Accumulating evidence demonstrates that the multifunctionality of TGF-beta1 is connected with the complexity of its cell signaling networks. TGF-beta1 signals through the interaction of type I and type II receptors to activate distinct intracellular pathways. Although the Smad signaling pathway is known as a canonical pathway induced by TGF-beta1, and has been the focus of many previous reviews, importantly TGF-beta1 also induces various Smad-independent signaling pathways. In this review, we describe evidence that supports current insights into the mechanism and function of TGF-beta-activated kinase 1 (TAK1), which has emerged as a critical signaling molecule in TGF-beta-induced Smad-independent signaling pathways. We also discuss the functional role of TAK1 in mediating the profibrotic effects of TGF-beta1.

Continuous hypoxia attenuates paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (1,1'-dimethyl-4,4'-bipyridinium dichloride; PQ), an effective and widely used herbicide, was commercially introduced in 1962. It is reduced by the electron donor NADPH, and then reduced PQ transfers the electrons to molecular oxygen, resulting in the production of reactive oxygen species (ROS), which are related to cellular toxicity. However, the influence of continuous hypoxia on PQ-induced ROS production has not fully been investigated. We evaluated in vitro the protective effect of continuous hypoxia on PQ-induced cytotoxicity in the human carcinogenic alveolar basal epithelial cell line (A549 cells) by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and live and dead assay, and by measuring lactate dehydrogenase (LDH) release. To elucidate the mechanism underlying this effect, we monitored the immunofluorescence of intracellular ROS and measured malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. Continuous hypoxia protected the A549 cells from PQ-induced cytotoxicity. Continuous hypoxia for a period of 24 h significantly reduced intracellular ROS, decreased MDA concentration in the supernatant, and normalized SOD and GPx activities. Continuous hypoxia attenuated PQ-induced cell toxicity in A549 cells. This protective effect might be attributable to the suppression of PQ-induced ROS generation.

Study on effect of fushenkeli on expression of TAK1 in human renal tubular epithelial cells and its possible mechanism / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effects of fushenkeli on the expression of TAK1 in the proliferation of the renal tubular epithelial cells induced by TGF-beta1 and its possible mechanism.</p><p><b>METHOD</b>Human renal tubular epithelial (HK-2) cells were divided into five groups:blank control group, TGF-beta1 group (5 microg x L(-1)), intervention group 1 (5 microg x L(-1) of TGF-beta1 + 100 mg x L(-1) of fushenkeli), intervention group 2 (5 microg x L(-1) of TGF-beta1 + 500 mg x L(-1) of fushenkeli) and intervention group 3 (5 microg x L(-1) of TGF-beta1 + 1 g x L(-1) of fushenkeli). HK-2 proliferation was detected by methyl thiazolyl tetrazolium (MTT) assay. Type IV collagen in the supernatants of the cultured HK-2 was detected by ELISA at 12, 24, 48 hours respectively. The protein and mRNA expressions of TAK1 was measured by Western blot and real-time quantitative PCR.</p><p><b>RESULT</b>1) The cell proliferation and the expression of type IV collagen were increased compared with the control group (P<0.05, P<0.01), but they were decreased in intervention group. 2) The expressions of protein and mRNA of TAK1 in TGF-beta1 group were upregulating significantly compared with control group (P<0.01), but they were downregulating in intervention group, especially in intervention group 3.</p><p><b>CONCLUSION</b>Fushenkeli could inhibits TAK1 expression induced by TGF-beta1 in the proliferation of HK-2 cell.</p>

Avaliação do padrão de crescimento na síndrome de Noonan em pacientes com mutações identificadas nos genes PTPN11, SOS1, RAF1 e KRAS / Growth pattern of patients with Noonan syndrome with identified mutations in PTPN11, SOS1, RAF1 e KRAS genes

A Síndrome de Noonan (SN) é caracterizada por baixa estatura proporcionada de início pós-natal, dismorfismos faciais, cardiopatia congênita e deformidade torácica. A frequência da SN é estimada entre 1:1000 e 1:2500 nascidos vivos, com distribuição semelhante em ambos os sexos. A herança é autossômica dominante com penetrância completa, porém a maioria dos casos é esporádica. Até o momento, mutações em genes da via RAS-MAPK (PTPN11, KRAS, SOS1, RAF1, MEK1, NRAS e SHOC2) foram identificadas em aproximadamente 70% dos pacientes. Uma das principais características fenotípicas da SN é a baixa estatura pós-natal, embora o mecanismo fisiopatológico do déficit de crescimento nesta síndrome ainda não esteja totalmente esclarecido. Estudos que avaliaram o padrão de crescimento linear em crianças com SN foram realizados anteriormente ao conhecimento do diagnóstico molecular dessa síndrome. No presente estudo, avaliamos a frequência de mutação nos genes PTPN11, SOS1, RAF1 e KRAS em 152 pacientes com SN e o padrão de crescimento linear (altura) e ponderal [índice de massa corpórea (IMC)] dos pacientes com mutação identificada. No total, mutações nos genes relacionados foram encontradas em 99 pacientes (65%) do nosso estudo, com predominância do gene PTPN11 (47%), seguido do SOS1 (9%), RAF1 (7%) e KRAS (3%). Foram construídas curvas específicas para SN de Altura e IMC para idade e sexo utilizando o método LMS. Os pacientes com SN apresentaram crescimento pré-natal preservado, porém o comprometimento do crescimento pós-natal foi observado desde o primeiro ano de vida, atingindo uma altura final de -2,5 e -2,2 desvios-padrão da média para população brasileira em homens e mulheres, respectivamente. O prejuízo da altura foi maior nos pacientes com mutação no gene RAF1 em comparação com os genes PTPN11 e SOS1. O IMC dos pacientes com SN apresentou queda de 1 desvio-padrão em relação à média da população brasileira normal. O comprometimento do IMC foi menor...

Noonan Syndrome (NS) is characterized by distinctive facial features, short stature and congenital heart defects. The estimated prevalence is 1:1000 to 1:2500 live births, affecting equally both sexes. It is an autosomal dominant disorder with complete penetrance, but most cases are sporadic. To date, mutations in the RAS/MAPK pathway genes (PTPN11, KRAS, SOS1, RAF1, MEK1, NRAS and SHOC2) were identified in approximately 70% of patients. One of the cardinal signs of NS is proportional postnatal short stature although the physiopathological mechanism of growth impairment remains unclear. The current knowledge about the natural history of growth associated with NS was described before molecular diagnosis era. In this study, we performed PTPN11, SOS1, RAF1, and KRAS mutation analysis in a cohort of 152 NS patients and studied the natural linear (height) and ponderal growth [body mass index (BMI)] of NS patients with related mutations. Mutations in NS-causative genes were found in 99 patients (65%) of our cohort. The most common mutated gene was PTPN11 (47%), followed by SOS1 (9%), RAF1 (7%) and KRAS (3%). Sex-specific percentile curves for height and BMI were constructed using the LMS method. NS patients had birth weight and length within normal ranges but the postnatal growth impairment was observed during the first year of life, reaching a final height of -2.3 and -2.2 standard deviations from the mean for Brazilian healthy men and women, respectively. Postnatal growth impairment was higher in RAF1 mutation patients than in patients with SOS1 and PTPN11 mutations. BMI values in NS patients were lower in comparison with normal Brazilian population. BMI values were higher in patients with RAF1 mutations than in patients with other genotypes. Patients with mutations in PTPN11 and SOS1 genes were more likely to have pulmonary valve stenosis, whereas hypertrophic cardiomyopathy was more common in patients with mutations in the gene RAF1...

Mechanism of inhibitory effect of P7 on 3T3 cell proliferation induced by basic fibroblast growth factor / 药学学报

To investigate the mechanism of inhibitory effect of a novel bFGF antagonist peptide isolated from the phage display random heptapeptide library on cell proliferation induced by basic fibroblast growth factor. The effect of P7 on cell morphology was observed under an inverted microscope. Flow cytometry was applied to analyze the effect of P7 on cell cycle progress of bFGF-stimulated cells. The effect of P7 on bFGF-induced activation of MEK and Erk1/2 in MAPK pathway was detected by Western blotting. The results showed that no significant cell morphology change was observed in the range of detected concentrations of P7. Cell cycle analysis showed that P7 decreased S-phase cell population and arrested cell cycle at the G0/G1 phase of bFGF-stimulated cells. The results of MAP kinase activation assay indicated that P7 decreased bFGF-induced MEK and Erk1/2 phosphorylation in a dose-dependent manner. P7 inhibited proliferation of bFGF-stimulated Balb/c 3T3 cells possibly via cell cycle arrest at the G0/G1 phase and down-regulation of signal molecular activation in MAPK pathway.

Regulation of the protein stability of POSH and MLK family

Sequential activation of the JNK pathway components, including Rac1/Cdc42, MLKs (mixed-lineage kinases), MKK4/7 and JNKs, plays a required role in many cell death paradigms. Those components are organized by a scaffold protein, POSH (Plenty of SH3's), to ensure the effective activation of the JNK pathway and cell death upon apoptotic stimuli. We have shown recently that the expression of POSH and MLK family proteins are regulated through protein stability. By generating a variety of mutants, we provide evidence here that the Nterminal half of POSH is accountable for its stability regulation and its over-expression-induced cell death. In addition, POSH's ability to induce apoptosis is correlated with its stability as well as its MLK binding ability. MLK family's stability, like that of POSH, requires activation of JNKs. However, we were surprised to find out that the widely used dominant negative (d/n) form of c-Jun could down-regulate MLK's stability, indicating that peptide from d/n c-Jun can be potentially developed into a therapeutical drug.

Epicatechin abolished TDCA-induced apoptosis in Huh7 cell by inhibiting Bax, p38 MAPK and ROS production / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the molecular mechanisms involved in anti-apoptotic effects of epicathechin in liver cells.</p><p><b>METHOD</b>Human hepatoma cell line (Huh7) was treated with 400 miromol x L(-1) taurodeoxycholic acid (TDCA) for 48 hours to induce apoptosis. Intracellular generation of reactive oxygen species (ROS) was detected with DCFH-DA assay. Caspase-3/7 activity was analyzed with EnzoLyte Homogeneous AMC kit. Cell proliferation was measured by MTT assay. The expression of Bax, Phospho-p38 MAPK and the levels of cytochrome C were assessed by Western-blot analysis.</p><p><b>RESULT</b>TDCA-dependent intracellular ROS production was 8-fold higher as compared to untreated cells, consequently resulting in 45% reduction of cell viability. Interestingly, pretreatment of cells with epicatechin resulted in a dose-dependent inhibition of TDCA-induced ROS generation and reduced cell apoptosis by threefold as compared to TDCA treatment alone. In addition epicatechin reduced Bax expression with consequential inhibition of cytochrome C release from mitochondria, inhibition of caspase 3/7 activation and p38 MAPK phosphorylation.</p><p><b>CONCLUSION</b>Epicatechin protects Huh7 cells from oxidative stress and mitochondria-induced apoptosis. The molecular mechanisms of anti-apoptotic effects of epicatechin were associated with inhibition of p38 MAPK phosphorylation and Bax expression, and reduction of ROS production. These findings implicate epicathechin might have potential as protective agent against a variety of oxidative stress-mediated liver conditions.</p>