Función biológica de AMPK- ß2 en músculo esquelético y su dinámica estructural después de la activación por SC4 / Biological role of AMPK- ß2 in skeletal muscle and its structural dynamics after activation by SC4

INTRODUCCIÓN: la Proteína Quinasa Activada por AMP (AMPK), es una enzima monitora y reguladora central del estado energético celular, por tanto, es responsable de la respuesta celular al suministro y demanda de energía. El AMP actúa como activador en condiciones de déficit energético, mientras que el ATP la inactiva cuando las condiciones energéticas son más favorables. Debido a su función central en el metabolismo, la AMPK surge como un blanco proteico prometedor para el tratamiento de diferentes enfermedades como la Diabetes Mellitus tipo 2 (DM2), Síndrome Metabólico (SM), Cáncer, entre otros. Existen múltiples isoformas de AMPK que se regulan y expresan diferencialmente en todo el organismo. La isoforma AMPK­ß2 se expresa casi exclusivamente en músculo esquelético y dado que este es el órgano primario para el almacenamiento y eliminación de Glucosa, AMPK­ß2 puede dirigir su homeostasis por una ruta independiente a la Insulina. La molécula activadora SC4 tiene una gran selectividad por AMPK­ß2 y debido a su función biológica, podría servir como modelo farmacológico para coadyuvar el tratamiento de enfermedades metabólicas. OBJETIVO: análisis de la dinámica molecular de activación de la AMPK­ß2. METODOLOGÍA: en el presente estudio, se emplean herramientas bioinformáticas como Chimera 1.15 y Phyton Molecular Viewer. RESULTADOS: el análisis in silico permitió comprender varios aspectos estructurales relacionados con la acción de SC4 sobre la estructura trimérica de la AMPK, los aminoácidos con los que interacciona y cómo su estructura química le otorga gran selectividad. También fue útil para en un futuro, ampliar los criterios de extracción, identificación y/o diseño de compuestos activos a partir de fuentes naturales, con propiedades funcionales similares o aún mejores a SC4, para así poder emplearlos con un enfoque terapéutico que beneficie a nuestra población.

INTRODUCTION: protein Kinase Activated by AMP (AMPK), is a monitor enzyme and a central regulator of the energetic cellular state, therefore, it is responsible for the cellular response to the supply and demand of energy. AMP acts as an activator in conditions of energy deficit, while ATP inactivates it when energy conditions are more favorable. Due to its central role in metabolism, AMPK appears as a promising protein target for the treatment of different diseases such as Diabetes Mellitus type 2 (DM2), Metabolic Syndrome (SM), and Cancer among others. There are multiple isoforms of AMPK that are regulated and differentially expressed throughout the body. The ß2-AMPK isoform is expressed almost exclusively in skeletal muscle and since this is the primary organ for Glucose disposal and storage, ß2-AMPK has an established role as a driver of insulin-independent Glucose clearance. The activator SC4 has a high selectivity for ß2-AMPK and due to its biological function; it could serve as a pharmacological model to aid the treatment of metabolic diseases. OBJETIVE: to analize the molecular dinamic of AMPK- ß2 activation. METHODOLOGY: in the present work we employed bioinformatics, Chimera 1.15 and Phyton Molecular Viewer. RESULTS: the in silico analysis allow us to understand many many structural features related to the action of SC4 on the trimeric structure of AMPK, the specific amino acids involved in the interaction and how its chemical structure gives it high selectivity. Thus, this structural analysis will be useful in order to broaden the criteria for extraction, identification and/or design of active compounds from natural sources, with similar or even better properties than SC4, to use them in a future, with a therapeutic approach that benefits our population.

Interações entre o relógio biológico e a sinalização do estado energético em plantas do complexo Saccharum / Biological clock and energy sensors in plants from Saccharum complex

A cana-de-açúcar e a cana energia são plantas intercruzáveis que compõe o complexo Saccharum. Estas plantas são fonte de biomassa para produção de açúcar, biocombustíveis, eletricidade, entre outros, e utilizam a energia assimilada pela fotossíntese de forma contrastante, ainda que ambas resultem em alta produtividade. O relógio biológico é um mecanismo molecular que gera informações sobre a hora do dia em conjunto com estímulos ambientais, adaptando respostas fisiológicas em prol de otimizar o desenvolvimento dos organismos em um ambiente cíclico, processo que regula cerca de 64% dos genes de cana-deaçúcar no campo. Em organismos sésseis como as plantas, o recorrente processo de produção de energia apenas durante o período luminoso, gera ritmos de metabólitos que influenciam na atividade de enzimas quinases que assim funcionam como sensores do estado energético, em vias conservadas nos eucariotos. Porém, pouco se sabe a respeito de como estes sinais são percebidos a nível transcricional, principalmente em plantas cultiváveis. Para elucidar como estas vias atuam em conjunto em plantas do complexo Saccharum, medimos o nível de transcrição de componentes do relógio biológico, de subunidades que compõe o complexo TOR, e da subunidade catalítica de SnRK1, KIN10. Medimos o desempenho do relógio biológico das variedades através da quantificação de amido em quatro pontos temporais, para obter uma dinâmica de produção e consumo, processo que é regulado pelo relógio biológico e tem genes com perfil de expressão rítmicos em cana de-açúcar. Curiosamente, uma das quatro variedades onde identificamos provável perfil rítmico de consumo de amido é a S.officinarum SP80-3280, cana-de-açúcar utilizada anteriormente para estudos de relógio biológico. Os nove acessos foram divididos em dois grupos com base em sua partição de carbono contrastante. HF (high fiber) com mais fibras e perfilho e grupo HS (high sucrose), com maior armazenamento de açúcares e amido que HF, em todos os horários de coleta, e com baixa produção de fibras. Estes grupos não diferem em expressão dos componentes de relógio biológico, no entanto, HS tem maior transcrição de uma subunidade do complexo TOR, em apenas um dos horários analisados (ZT12). Em conjunto, a expressão dos componentes do relógio biológico divide os acessos entre os que possuem altos níveis de transcrição de ScLHY, no ZT03, e os que possuem maior transcrição dos genes PRR59, 73 e 95, no ZT12, grupos com contrastante partição de carbono. A transcrição dos sensores energéticos se correlaciona no começo da noite em acessos de HS e Krakatau e, no começo da manhã, em acessos de HF e IN84-105, sem agrupar as variedades por espécie ou destino de carbono. Este trabalho sugere que há diferentes níveis de correlação entre a transcrição dos genes mensurados e as contrastantes partições de carbono das plantas do complexo Saccharu

Sugarcane and Energycane are intercrossable plants that make up the Saccharum complex. These plants are a source of biomass, sugar, biofuels, electricity among others, and even though they use the energy assimilated by photosynthesis in a contrasting way, both results in high productivity. The biological clock is a molecular mechanism that generates information about the time of day in conjunction with environmental stimuli, adapting physiological responses to optimize the development of organisms in a cyclic environment, a process that regulates about 64% of sugarcane genes in field-grown plants. In organisms such as plants, the recurrent process of energy production that happens only during the luminous period generates rhythmicity that may influence the activity of kinase enzymes, thus giving an energy sensor property for then. However, little is known about how these signs are perceived at the transcriptional level, especially in crops and monocots. To elucidate how these pathways act together in plants of the Saccharum complex, we measured the transcription level of the daytime loop of the biological clock, subunits that make up the TOR complex, and the catalytic subunit of SnRK1, KIN10. We measured starch content in four time points, to obtain a dynamic of production and consumption, a process that is regulated by the biological clock and has genes with a rhythmic expression profile in sugarcane. Interestingly, one of the four varieties where we could identify a probable rhythmic profile of starch consumption is a sugarcane SP80-3280 (S. officinarum), that have been used for biological clock studies. The nine genotypes were divided into two groups based on their contrasting carbon partition. HF (high fiber) with more fiber and tiller and group HS (high sucrose), with higher sugar and starch storage than HF, but with lower fiber production. These groups do not differ in expression of biological clock components; however, HS has a higher transcription of a subunit of the TOR complex, in only one of the analyzed times (ZT12). Together, the expression of components of the biological clock divides the genotypes between those with higher levels of ScLHY in ZT03 and those with more transcripts of PRR59, 73 and 95 genes in ZT12, groups that also have contrasting carbon partition. The transcription of TOR complex correlates in the early evening in HS and KRAKATAU, but in the morning, in HF and IN84-105, with no clear correlation with the C destination preferences. This work suggests that there are different levels of correlation between the transcription of biological clock and energy sensors component genes and the contrasting carbon partitions of plants from the Saccharum complex

O receptor aril de hidrocarbonetos (AhR) em melanomas: associação com mutações BRAF, com a progressão da doença e identificação de ligantes / The aryl hydrocarbon receptor (AhR) in melanomas: association with BRAF mutations, with disease progression and ligand identification

O melanoma é um tipo de câncer de pele geneticamente diverso, que surge diante das transformações em melanócitos. A mutação BRAFV600E está presente em mais de 90% de todas as mutações em BRAF, sendo assim ocorre em cerca de 50% dos casos registrados. As mutações em NRAS, ocupam o segundo lugar entre as mutações mais prevalentes, cerca de 20% dos casos. Informações sobre as assinaturas genéticas, permitiram o desenvolvimento de terapia alvo dirigida. O Vemurafenib, inibidor da quinase BRAFV600E, apresentou inicialmente resultados bastante satisfatórios, contudo existe registro de casos de recidiva e resistência. O receptor aril de hidrocarbonetos é expresso em vários componentes da pele, e assim está relacionado a homeostase e fisiopatologia da pele. Diante disso, a avaliação da expressão do receptor em um painel de linhagens mutadas para NRAS e BRAF, e BRAF resistentes, mostrou-se maior do que a encontrada em melanócitos. Também encontramos maior expressão de mRNA de AhR em linhagens de melanoma derivadas de sítio primário e metastático, mutadas para BRAFV600E, quando comparadas ao melanócito. Agregado a isto, a análise in silico no TCGA (The Cancer Genome Atlas) mostrou que há 18% de alteração genética em AhR, sendo em maior parte a alta regulação de mRNA. Também, a análise do banco público GSE12391, mostrou aumento de mRNA de AhR na fase de crescimento vertical do melanoma. Assim, concluímos que há maior expressão de mRNA e sua importância nas fases de desenvolvimento do melanoma, tanto nos processos iniciais quanto em processos de migração, invasão e metástase. Ainda, encontramos maior mRNA do receptor em linhagens resistentes ao Vemurafenib. Este resultado sustenta a hipótese de que AhR pode ser considerado um marcador de resistência em melanomas. O AhR, inicialmente no citoplasma, quando ativado pode atuar como fator de transcrição regulando vários genes que apresentam sequências definidas, participando de respostas carcinogênicas. Compostos halogenados e moléculas endógenas derivadas das vias de metabolização do triptofano são agonistas do receptor. Anteriormente, nosso grupo mostrou que linhagens de melanoma incubadas com triptamina e DMT exibiram menor clonogenicidade. Diante de uma literatura escassa sobre o papel do DMT no melanoma e com base nestes resultados, nosso objetivo foi avaliar o papel de AhR nesta interface DMT-melanoma. Para isto, nosso objetivo foi construir linhagem editada geneticamente para AhR através da ferramenta CRISPR-Cas9. Vários foram os esforços, sem sucesso, utilizados nas tentativas de comprovar a manutenção de células editadas na cultura. Atrelamos a este resultado a possibilidade de haver duas subpopulações editadas geneticamente pós CRISPR-Cas9, onde uma destas manteve o padrão de crescimento semelhante às células wild type. Devido a este crescimento diferencial, não obtivemos congruências nos ensaios e postulamos a perda do possível nocaute. A partir disso, realizamos ensaios de interactoma para avaliar a interação de DMT-AhR. Nosso resultado sugere a interação de DMT ao receptor sigma 1, e não ao receptor aril de hidrocarbonetos. Desta forma, o interactoma sustenta a hipótese de que DMT não é um ligante de AhR. Para certificar este resultado análises de docking associados a ensaios biológicos, avaliando o papel do receptor, devem ser realizados para averiguar a afinidade e seletividade de DMT como ligante do receptor na linhagem de melanoma

Melanoma is a genetically diverse type of skin cancer, which arises from changes in melanocytes. The BRAFV600E mutation is present in more than 90% of all BRAF mutations, so it occurs in about 50% of registered cases. Mutations in NRAS occupy the second place among the most prevalent mutations, about 20% of cases. Information on genetic signatures allowed the development of targeted therapy. vemurafenib, kinase inhibitor BRAFV600E, initially presented very satisfactory results, however there is a record of cases of relapse and resistance. The aryl hydrocarbon receptor is expressed in several components of the skin and is thus related to homeostasis and skin pathophysiology. Therefore, the evaluation of receptor expression in a panel of strains mutated to NRAS and BRAF, and resistant BRAF, proved to be greater than that found in melanocytes. We also found main expression of AhR mRNA in melanoma strains derived from primary and metastatic site, mutated to BRAFV600E, when compared to melanocyte. Added to this, the in silico analysis in TCGA (The Cancer Genome Atlas) showed that there is 18% of genetic alteration in AhR, being mostly the high regulation of mRNA. Also, an analysis by the public bank GSE12391, showed an increase in AhR mRNA in the vertical growth phase of melanoma. Thus, it is concluded that there is greater expression of mRNA and its importance in the stages of development of melanoma, both in recent processes and in the processes of migration, invasion and metastasis. In addition, we found higher receptor mRNA in strains resistant to vemurafenib. This result supports the hypothesis that AhR can be considered a marker of resistance in melanomas. AhR, initially in the cytoplasm, when activated can act as a transcription factor regulating several genes that have defined sequences, participating in carcinogenic responses. Along with this, we show that along the tumor progression, there is an increase in AhR in the radial growth phase of melanoma. Halogenated compounds and endogenous molecules derived from the tryptophan metabolism pathways are receptor agonists. Previously, our group showed that melanoma strains incubated with tryptamine and DMT exhibited less clonogenicity. In view of a scarce literature on the role of DMT in melanoma and based on these results, our objective was to evaluate the role of AhR in this DMT-melanoma interface. For this, our goal was to build genetically edited strain for AhR using the CRISPR-Cas9 tool. Several efforts were unsuccessful in attempts to prove the maintenance of cells edited in the culture. We linked to this result the possibility of having two subpopulations genetically edited after CRISPR-Cas9, where one of them maintained the growth pattern like wild type cells. Due to this differential growth, we did not obtain congruence in the tests and postulated the loss of the possible knockout. From that, we performed interactome assays to evaluate the DMT-AhR interaction. Our result suggests the interaction of DMT with the sigma 1 receptor, and not the aryl hydrocarbon receptor. Thus, the interactome supports the hypothesis that DMT is not an AhR ligand. To certify this result, docking analyses associated with biological assays, evaluating the role of the receptor, should be performed to ascertain the affinity and selectivity of DMT as a ligand of the receptor in the melanoma lineage

Lupine as a potential agent against diet-induced obesity through adenosine monophosphate-activated protein kinase pathway: crude oil versus nanoemulsion formulations

Obesity represents a major challenge to the pharmaceutical community due to the minimal availability of anti-obesity drugs and the drawbacks of current weight-loss agents. The study described herein presents lupine oil, in two pharmaceutical formulations, as a potential anti-obesity agent via its effect on different physiological, biochemical, and hormonal parameters. Rats were divided into two groups; one group was continued on a standard commercial rodent diet and served as the non-obese control. The other group was fed a high-fat diet for 7 weeks to prepare an obese rat model. Then, the obese rats were divided into groups to receive 100 mg/kg of the crude lupine oil or nanoemulsion for 10 or 20 days. Lupine oil showed a potent body weight-reducing effect and improved insulin resistance. The oil altered obesity-induced hyperlipidemia and it enhanced the leptin/adiponectin/AMPK hormonal system in epididymal fat, serum, and liver, to which all the above physiological activities could be attributed. The nanoemulsion formulation of lupine oil significantly amplified the activity for all the above physiological and hormonal parameters when compared to the crude oil formulation. Lupine oil nanoemulsion could be used as a potential drug against diet-induced obesity.

Characteristics of Renal Cell Carcinoma Harboring TPM3-ALK Fusion

The World Health Organization 2016 edition assigned anaplastic lymphoma kinase (ALK) rearrangement-associated renal cell carcinoma (ALK-RCC) as an emerging renal tumor entity. Identifying ALK-RCC is important because ALK inhibitors have been shown to be effective in treatment. Here, we report the case of a 14-year-old young man with ALK-RCC. Computed tomography revealed a well-demarcated 5.3-cm enhancing mass at the upper pole of the left kidney. There was no further history or symptoms of the sickle-cell trait. The patient underwent left radical nephrectomy. Pathologically, the mass was diagnosed as an unclassified RCC. Targeted next-generation sequencing identified a TPM3-ALK fusion gene. The present report and literature review demonstrate that TPM3-ALK RCC may be associated with distinct clinicopathological features. Microscopically, the tumors showed diffuse growth and tubulocystic changes with inflammatory cell infiltration. Tumor cells were dis-cohesive and epithelioid with abundant eosinophilic cytoplasm and cytoplasmic vacuoles. If morphological features and TFE3 expression are present in adolescent and young patients, molecular tests for ALK translocation should be performed. This awareness is critically important, because ALK rearrangement confers sensitivity to ALK inhibitors.

Current Status of Immunotherapy for Lung Cancer and Future Perspectives / 결핵

Correlation of PD-L1 Expression Tested by 22C3 and SP263 in Non-Small Cell Lung Cancer and Its Prognostic Effect on EGFR Mutation–Positive Lung Adenocarcinoma / 결핵

Evidence for the Presence of Long-Lived Plasma Cells in Nasal Polyps

PURPOSE: Plasma cells and immunoglobulins (Igs) play a pivotal role in the induction and maintenance of chronic inflammation in nasal polyps. During secondary immune responses, plasma cell survival and Ig production are regulated by the local environment. The purpose of the present study was to investigate the presence of long-lived plasma cells (LLPCs) and specific survival niches for LLPCs in human nasal polyps.METHODS: Nasal mucosal samples were cultured with an air-liquid interface system and the Ig levels in culture supernatants were analyzed by enzyme-linked immunosorbent assay. The characteristics of LLPCs in nasal polyps were determined by immunohistochemistry and immunofluorescence. The expression of neurotrophins as well as their receptors was detected by quantitative real-time polymerase chain reaction, immunohistochemistry, immunofluorescence, and Western blotting.RESULTS: The numbers of CD138⁺ total plasma cells and BCL2⁺ plasma cells were increased in both eosinophilic and non-eosinophilic nasal polyps compared with those in normal tissues. The production of IgG, IgA, and IgE was detected in culture supernatants even after a 32-day culture of nasal polyps. Although the total numbers of plasma cells were decreased in nasal polyps after culture, the numbers of BCL2⁺ plasma cells remained stable. The expression of nerve growth factor (NGF) as well as tropomyosin receptor kinase (Trk) A, a high-affinity receptor for NGF, was upregulated in both eosinophilic and non-eosinophilic nasal polyps. In addition, BCL2⁺ plasma cell numbers were positively correlated with NGF and TrkA mRNA expression in nasal mucosal tissues. Polyp plasma cells had the expression of TrkA.CONCLUSIONS: Human nasal polyps harbor a population of LLPCs and NGF may be involved in their prolonged survival. LLPCs may be a novel therapeutic target for suppressing the local Ig production in nasal polyps.

Epithelial PI3K-δ Promotes House Dust Mite-Induced Allergic Asthma in NLRP3 Inflammasome-Dependent and -Independent Manners

PURPOSE: Phosphoinositide 3-kinase (PI3K)-δ-dependent Akt activation is known to play critical roles in various immune responses of white blood cells in which PI3K-δ isoform is mostly expressed in contrast to the classes IA PI3Ks p110α and p110β. However, the immunological role of PI3K-δ isoform is still controversial in airway epithelium under house dust mite (HDM)-induced allergic response. This study aimed to evaluate the role of PI3K-δ isoform in HDM-induced allergic responses, focusing on NLRP3 inflammasome activation in airway epithelium.METHODS: We used wild-type mice and PI3K-δ knock-out (KO) mice for HDM-induced asthma animal model and also performed in vitro experiments using primary cultured murine tracheal epithelial cells and human airway epithelial cells.RESULTS: PI3K-δ activated HDM-induced NLRP3 inflammasome and epithelial cell-derived cytokines in the lung including airway epithelial cells. PI3K-δ KO mice or knock-down of PI3K-δ using siRNA exhibited the significant reduction in allergic asthmatic features and the suppression of NLRP3 inflammasome assembly as well as epithelial cell-derived cytokines. Interestingly, significantly increased expression of PI3K-δ isoform was observed in stimulated airway epithelial cells and the increases in epithelial cell-derived cytokines were markedly suppressed by blocking PI3K-δ, while these cytokine levels were independent of NLRP3 inflammasome activation.CONCLUSIONS: The results of this study suggest that PI3K-δ-isoform can promote HDM-induced allergic airway inflammation via NLRP3 inflammasome-dependent response as well as via NLRP3 inflammasome-independent epithelial cell activation.

Therapeutic Effect of a Novel Chimeric Molecule Targeting Both Somatostatin and Dopamine Receptors on Growth Hormone-Secreting Pituitary Adenomas

BACKGROUND: Acromegaly is a rare disease primarily caused by growth hormone (GH)-secreting pituitary adenomas, and its treatment is costly. Moreover, some patients are unresponsive to treatment. Hence, there are increasing efforts to develop new drugs with improved effectiveness for this disease. BIM23B065 is a novel chimeric molecule that acts on both somatostatin and dopamine receptors. This study aimed to investigate the effects of BIM23B065 compared with those of a somatostatin receptor analog and a dopamine agonist.METHODS: The effects of BIM23B065 on the proliferation, GH and insulin-like growth factor-1 (IGF-1) levels, and extracellular signal-regulated kinase (ERK) 1/2 and cyclic AMP response element binding (CREB) phosphorylation of GH3 cells were investigated with MTS assay, enzyme-linked immunosorbent assay, and Western blotting, respectively. The dosage and treatment duration of BIM23B065 were tested in animal models of GH-secreting pituitary adenoma. The effect of BIM23B065 (3 mg/kg/day) on changes in IGF-1 levels before and after treatment was further investigated.RESULTS: In vitro, BIM23B065 treatment decreased GH release in the culture media and downregulated ERK 1/2 and CREB phosphorylation to 22% and 26%, respectively. In vivo, IGF-1 expression decreased to 50 % after 4 weeks of treatment with BIM23B065 using an osmotic pump implant. Moreover, magnetic resonance imaging results showed that the tumor size decreased significantly following treatment with BIM23B065 for 4 weeks.CONCLUSION: The novel chimeric molecule was effective in decreasing IGF-1 and GH levels and may serve as an effective therapeutic agent for acromegaly.

Neuroprotective potential of imatinib in global ischemia-reperfusion-induced cerebral injury: possible role of Janus-activated kinase 2/signal transducer and activator of transcription 3 and connexin 43

The present study was aimed to explore the neuroprotective role of imatinib in global ischemia-reperfusion-induced cerebral injury along with possible mechanisms. Global ischemia was induced in mice by bilateral carotid artery occlusion for 20 min, which was followed by reperfusion for 24 h by restoring the blood flow to the brain. The extent of cerebral injury was assessed after 24 h of global ischemia by measuring the locomotor activity (actophotometer test), motor coordination (inclined beam walking test), neurological severity score, learning and memory (object recognition test) and cerebral infarction (triphenyl tetrazolium chloride stain). Ischemia-reperfusion injury produced significant cerebral infarction, impaired the behavioral parameters and decreased the expression of connexin 43 and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) in the brain. A single dose administration of imatinib (20 and 40 mg/kg) attenuated ischemia-reperfusion-induced behavioral deficits and the extent of cerebral infarction along with the restoration of connexin 43 and p-STAT3 levels. However, administration of AG490, a selective Janus-activated kinase 2 (JAK2)/STAT3 inhibitor, abolished the neuroprotective actions of imatinib and decreased the expression of connexin 43 and p-STAT3. It is concluded that imatinib has the potential of attenuating global ischemia-reperfusion-induced cerebral injury, which may be possibly attributed to activation of JAK2/STAT3 signaling pathway along with the increase in the expression of connexin 43.

Ezrin-radixin-moesin proteins are regulated by Akt-GSK3β signaling in the rat nucleus accumbens core

The ezrin-radixin-moesin (ERM) proteins are a family of membrane-associated proteins known to play roles in cell-shape determination as well as in signaling pathways. We have previously shown that amphetamine decreases phosphorylation levels of these proteins in the nucleus accumbens (NAcc), an important neuronal substrate mediating rewarding effects of drugs of abuse. In the present study, we further examined what molecular pathways may be involved in this process. By direct microinjection of LY294002, a PI3 kinase inhibitor, or of S9 peptide, a proposed GSK3β activator, into the NAcc core, we found that phosphorylation levels of ERM as well as of GSK3β in this site are simultaneously decreased. These results indicate that ERM proteins are under the regulation of Akt-GSK3β signaling pathway in the NAcc core. The present findings have a significant implication to a novel signal pathway possibly leading to structural plasticity in relation with drug addiction.

Overexpression of HER2/HER3 and clinical feature of ovarian cancer / 부인종양

OBJECTIVES: Human epidermal growth factor receptor-2 (HER2) and 3 (HER3) belong to the epidermal growth factor receptor (EGFR) family of transmembrane receptor tyrosine kinases. In this study, we assessed HER2/HER3 expression levels in specimens of epithelial ovarian cancer and determined their correlation with clinical features of ovarian cancer. METHODS: Tissue microarrays (TMAs) were prepared from paraffin blocks of 105 ovarian tumour samples. HER2, HER3, PI3K, Akt, p-Akt, mTOR, p-mTOR, S6, and p-S6 expression levels were investigated using immunohistochemistry (IHC). HER2 and HER3 amplifications were determined using in situ hybridization (ISH). The correlation between HER2/3 expression and disease outcome of the patients including surgical outcome, progression-free survival (PFS) and overall survival (OS) was analysed. RESULTS: HER2 positivity was 3.8% by IHC and 5.7% by ISH, whereas that of HER3 was 12.4% and 8.6%, respectively. HER2 status by either IHC or ISH was not related to PFS (p=0.128, 0.168, respectively) and OS (p=0.245, 0.164, respectively). However, the HER3 status determined using fluorescence ISH was associated with poor PFS (p=0.035 on log rank test), which was a significant risk factor even after adjusting other possible risk factors in multivariate analysis (hazard ratio=2.377 [1.18–7.49], p=0.021). Expressions of Akt, p-mTOR, and S6 were also related with poor progression (p=0.008, 0.049, 0.014, respectively). CONCLUSION: HER3 is possibly an independent marker for poor prognosis in individuals with ovarian cancer, as the HER3 signalling pathway is distinct from that of HER2. The possibility of targeted therapy for patients with HER3 alteration in ovarian cancer should be evaluated.

Protective effect of Cordyceps militaris against hydrogen peroxide-induced oxidative stress in vitro

BACKGROUND/OBJECTIVES: Excessive production of reactive oxygen species (ROS) such as hydroxyl (·OH), nitric oxide (NO), and hydrogen peroxide (H2O2) is reported to induce oxidative stress. ROS generated by oxidative stress can potentially damage glial cells in the nervous system. Cordyceps militaris (CM), a kind of natural herb widely found in East Asia. In this study, we investigated the free radical scavenging activity of the CM extract and its neuroprotective effects in H2O2-induced C6 glial cells. MATERIALS/METHODS: The ethanol extract of CM (100–1,000 µg/mL) was used to measure DPPH, ·OH, and NO radical scavenging activities. In addition, hydrogen peroxide (H2O2)-induced C6 glial cells were treated with CM at 0.5–2.5 µg/mL for measurement of cell viability, ROS production, and protein expression resulting from oxidative stress. RESULTS: The CM extract showed high scavenging activities against DPPH, ·OH, and NO radicals at concentration of 1,000 µg/mL. Treatment of CM with H2O2-induced oxidative stress in C6 glial cells significantly increased cell viability, and decreased ROS production. Cyclooxygenase-2 and inducible nitric oxide synthase protein expression was down-regulated in CM-treated groups. In addition, the protein expression level of phospho-p38 mitogen-activated protein kinase (p-p38 MAPK), phospho-c-Jun N-terminal kinase (p-JNK), and phospho-extracellular regulated protein kinases (p-ERK) in H2O2-induced C6 glial cells was down-regulated upon CM administration. CONCLUSION: CM exhibited radical scavenging activity and protective effect against H2O2 as indicated by the increased cell viability, decreased ROS production, down-regulation of inflammation-related proteins as well as p-p38, p-JNK, and p-ERK protein levels. Therefore, we suggest that CM could play the protective role from oxidative stress in glial cells.

Mannosylerythritol lipids ameliorate ultraviolet A-induced aquaporin-3 downregulation by suppressing c-Jun N-terminal kinase phosphorylation in cultured human keratinocytes

Mannosylerythritol lipids (MELs) are glycolipids and have several pharmacological efficacies. MELs also show skin-moisturizing efficacy through a yet-unknown underlying mechanism. Aquaporin-3 (AQP3) is a membrane protein that contributes to the water homeostasis of the epidermis, and decreased AQP3 expression following ultraviolet (UV)-irradiation of the skin is associated with reduced skin moisture. No previous study has examined whether the skin-moisturizing effect of MELs might act through the modulation of AQP3 expression. Here, we report for the first time that MELs ameliorate the UVA-induced downregulation of AQP3 in cultured human epidermal keratinocytes (HaCaT keratinocytes). Our results revealed that UVA irradiation decreases AQP3 expression at the protein and messenger RNA (mRNA) levels, but that MEL treatment significantly ameliorated these effects. Our mitogen-activated protein kinase inhibitor analysis revealed that phosphorylation of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, mediates UVA-induced AQP3 downregulation, and that MEL treatment significantly suppressed the UVA-induced phosphorylation of JNK. To explore a possible mechanism, we tested whether MELs could regulate the expression of peroxidase proliferator-activated receptor gamma (PPAR-γ), which acts as a potent transcription factor for AQP3 expression. Interestingly, UVA irradiation significantly inhibited the mRNA expression of PPAR-γ in HaCaT keratinocytes, whereas a JNK inhibitor and MELs significantly rescued this effect. Taken together, these findings suggest that MELs ameliorate UVA-induced AQP3 downregulation in HaCaT keratinocytes by suppressing JNK activation to block the decrease of PPAR-γ. Collectively, our findings suggest that MELs can be used as a potential ingredient that modulates AQP3 expression to improve skin moisturization following UVA irradiation-induced damage.

Discovery of an Indirubin Derivative as a Novel c-Met Kinase Inhibitor with In Vitro Anti-Tumor Effects

The c-Met protein is a receptor tyrosine kinase involved in cell growth, proliferation, survival, and angiogenesis of several human tumors. Overexpression of c-Met has been found in gastric cancers and correlated with a poor prognosis. Indirubin is the active component of Danggui Longhui Wan, which is a traditional Chinese antileukemic recipe. In the present study, we tested the anti-cancer effects of an indirubin derivative, LDD-1937, on human gastric cancer cells SNU-638. When we performed the in vitro kinase assay against the c-Met activity, LDD-1937 inhibited the activity of c-Met. This result was confirmed by immunoblot and immunofluorescence of phosphorylated c-Met. Immunoblot analysis showed that LDD-1937 decreased the expression of the Erk1/2, STAT3, STAT5, and Akt, downstream proteins of c-Met. In addition, LDD-1937 reduced the cell viability and suppressed colony formation and migration of SNU-638 cells. Furthermore, LDD-1937 induced G2/M phase arrest in the SNU-638 cells by decreasing the expression levels of cyclin B1 and CDC2. Cleaved-PARP, an apoptosis-related protein, was up-regulated in cells treated with LDD-1937. Overall, this study suggests that LDD-1937 may be a novel small-molecule with therapeutic potential for selectively inhibiting c-Met and c-Met downstream pathways in human gastric cancers overexpressing c-Met.

Amitriptyline inhibits the MAPK/ERK and CREB pathways and proinflammatory cytokines through A3AR activation in rat neuropathic pain models / 대한마취과학회지

BACKGROUND: The pain-relief properties of tricyclic antidepressants can be attributed to several actions. Recent observations suggest that adenosine is involved in the antinociceptive effect of amitriptyline. The A3 adenosine receptor (A3AR) is the only adenosine subtype overexpressed in inflammatory and cancer cells. This study was performed to investigate the role of A3AR in the anti-nociceptive effect of amitriptyline. METHODS: Spinal nerve-ligated neuropathic pain was induced by ligating the L5 and L6 spinal nerves of male Sprague-Dawley rats. The neuropathic rats were randomly assigned to one of the following three groups (8 per group): a neuropathic pain with normal saline group, a neuropathic pain with amitriptyline group, and a neuropathic pain with amitriptyline and 3-ethyl-5-benzyl- 2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS) group. Amitriptyline or saline was administered intraperitoneally and 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS-1191), an A3AR antagonist, was injected subcutaneously immediately before amitriptyline administration. The level of extracellular signal-regulated kinase P44/42 (ERK1/2), cyclic AMP response element-binding protein (CREB), and proinflammatory cytokines were assessed using immunoblotting or reverse-transciption polymerase chain reaction. RESULTS: Amitriptyline increased the mechanical withdrawal threshold of the neuropathic rats. The level of phospho-ERK1/2 and phospho-CREB proteins, and proinflammatory cytokines produced by spinal nerve ligation were significantly reduced by amitriptyline administration. However, the use of MRS-1191 before amitriptyline administration not only reduced the threshold of mechanical allodynia, but also increased the signaling protein and proinflammatory cytokine levels, which were reduced by amitriptyline. CONCLUSIONS: The results of this study suggest that the anti-nociceptive effect of amitriptyline involves the suppression of ERK1/2 and CREB signaling proteins, and A3AR activation also affects the alleviation of the inflammatory response.

Expression of polo-like kinase 1 in pre-implantation stage murine somatic cell nuclear transfer embryos

Somatic cell nuclear transfer (SCNT) has various applications in research, as well as in the medical field and animal husbandry. However, the efficiency of SCNT is low and the accurate mechanism of SCNT in murine embryo development is unreported. In general, the developmental rate of SCNT murine embryos is lower than in vivo counterparts. In previous studies, polo-like kinase 1 (Plk1) was reported to be a crucial element in cell division including centrosome maturation, cytokinesis, and spindle formation. In an initial series of experiments in this study, BI2536, a Plk1 inhibitor, was treated to in vivo-fertilized embryos and the embryos failed to develop beyond the 2-cell stage. This confirmed previous findings that Plk1 is crucial for the first mitotic division of murine embryos. Next, we investigated Plk1's localization and intensity by immunofluorescence analysis. In contrast to normally developed embryos, SCNT murine embryos that failed to develop exhibited two types of Plk1 expressions; a low Plk1 expression pattern and ectopic expression of Plk1. The results show that Plk1 has a critical role in SCNT murine embryos. In conclusion, this study demonstrated that the SCNT murine embryos fail to develop beyond the 2-cell stage, and the embryos show abnormal Plk1 expression patterns, which may one of the main causes of developmental failure of early SCNT murine embryos.

Cyclosporin A aggravates hydrogen peroxide-induced cell death in kidney proximal tubule epithelial cells / 대한해부학회지

Cyclosporin A (CsA) does not only exert a toxic effect on kidney parenchymal cells, but also protects them against necrotic cell death by inhibiting opening of mitochondrial permeability transition pore. However, whether CsA plays a role in hydrogen peroxide-induced kidney proximal tubular cell death is currently unclear. In the present study, treatment with CsA further increased apoptosis and necrosis in HK-2 human kidney proximal tubule epithelial cells during exposure to hydrogen peroxide. In addition, hydrogen peroxide-induced p53 activation and BH3 interacting-domain death agonist (BID) expression were higher in CsA-treated cells than those in non-treated cells, whereas hydrogen peroxide-induced activation of mitogen-activated protein kinases including p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase and activation of protein kinase B were not significantly altered by treatment with CsA. In oxidant-antioxidant system, reactive oxygen species (ROS) production induced by hydrogen peroxide was further enhanced by treatment with CsA. However, expression levels of antioxidant enzymes including manganese superoxide dismutase, copper/zinc superoxide dismutase, and catalase were not altered by treatment with hydrogen peroxide or CsA. Treatment with CsA further enhanced mitochondrial membrane potential induced by exposure to hydrogen peroxide, although it did not alter endoplasmic reticulum stress based on expression of glucose-regulated protein 78 and 94. Taken together, these data suggest that CsA can aggravate hydrogen peroxide-induced cell death through p53 activation, BID expression, and ROS production.

Induction of Melanoma Cell-Selective Apoptosis Using Anti-HER2 Antibody-Conjugated Gold Nanoparticles

PURPOSE: This study was conducted to verify the induction and mechanism of selective apoptosis in G361 melanoma cells using anti-HER2 antibody-conjugated gold nanoparticles (GNP-HER2). MATERIALS AND METHODS: Following GNP-HER2 treatment of G361 cells, cell cycle arrest and apoptosis were measured by WST-1 assay, Hemacolor staining, Hoechst staining, immunofluorescence staining, fluorescence-activated cell sorting analysis, and Western blotting.