The role of TMEM26 in disrupting tight junctions and activating NF-κB signaling to promote epithelial-mesenchymal transition in esophageal squamous cell carcinoma.

OBJECTIVES: Metastasis is one of the biggest challenges in the management of Esophageal Squamous Cell Carcinoma (ESCC), of which molecular mechanisms remain elusive. The present study aimed to explore the roles and underlying mechanisms of Transmembrane protein 26 (TMEM26) in ESCC. METHOD: TMEM26 expressions in tumorous and adjacent tissues from patients with ESCC and in normal esophageal epithelial and ESCC cell lines were detected by immunostaining and western blotting, respectively. The Epithelial-Mesenchymal Transition (EMT), a critical process during metastasis, was investigated by wound healing and Transwell assays, and EMT-related proteins were examined after the TMEM26 alteration in ESCC cell lines. NF-κB signaling activation and Tight Junction (TJ) protein expression were analyzed by western blotting and immunofluorescence, respectively. In vivo verification was performed on the liver metastatic murine model. RESULTS: Compared with non-cancerous esophageal tissues and cells, the TMEM26 expression level was higher in ESCC samples and cell lines, where the plasma membrane localization of TMEM26 was observed. The EMT-related processes of ESCC cells were suppressed by RNAi depletion of TMEM26 but aggravated by TMEM26 overexpression. Mechanistically, TMEM26 promoted NF-κB signaling to accelerate EMT in ESCC cells. The plasma membrane presentation and assembly of TJ proteins were impaired by TMEM26. CONCLUSION: Overall, TMEM26 acts as a critical determinant for EMT in ESCC cells by disrupting TJ formation and promoting NF-κB signaling, which may be a potential therapeutic target for treating metastatic ESCC.

The role of TMEM26 in disrupting tight junctions and activating NF-κB signaling to promote epithelial-mesenchymal transition in esophageal squamous cell carcinoma

Abstract Objectives Metastasis is one of the biggest challenges in the management of Esophageal Squamous Cell Carcinoma (ESCC), of which molecular mechanisms remain elusive. The present study aimed to explore the roles and underlying mechanisms of Transmembrane protein 26 (TMEM26) in ESCC. Method TMEM26 expressions in tumorous and adjacent tissues from patients with ESCC and in normal esophageal epithelial and ESCC cell lines were detected by immunostaining and western blotting, respectively. The Epithelial-Mesenchymal Transition (EMT), a critical process during metastasis, was investigated by wound healing and Transwell assays, and EMT-related proteins were examined after the TMEM26 alteration in ESCC cell lines. NF-κB signaling activation and Tight Junction (TJ) protein expression were analyzed by western blotting and immunofluorescence, respectively. In vivo verification was performed on the liver metastatic murine model. Results Compared with non-cancerous esophageal tissues and cells, the TMEM26 expression level was higher in ESCC samples and cell lines, where the plasma membrane localization of TMEM26 was observed. The EMT-related processes of ESCC cells were suppressed by RNAi depletion of TMEM26 but aggravated by TMEM26 overexpression. Mechanistically, TMEM26 promoted NF-κB signaling to accelerate EMT in ESCC cells. The plasma membrane presentation and assembly of TJ proteins were impaired by TMEM26. Conclusion Overall, TMEM26 acts as a critical determinant for EMT in ESCC cells by disrupting TJ formation and promoting NF-κB signaling, which may be a potential therapeutic target for treating metastatic ESCC.

Thrombopoietin is associated with a prognosis of gastric adenocarcinoma

SUMMARY OBJECTIVE Thrombopoietin (THPO) is well-known as a megakaryocyte growth and development factor (MGDF) involved in megakaryocyte proliferation and maturation. To explore the biological effects of THPO in gastric adenocarcinoma, we conducted this study. Methods: By accessing the TCGA database, the expression level of THPO was determined in tumor tissues. The association between THPO expression and clinical features, or prognostic significance was described by Cox regression analysis and Kaplan-Meier. The SiRNA method was used to decline the THPO expression; then cell viability, invasion, and migration were detected to verify the effects of the knockdown of THPO. qPCR and western blotting were implemented to examine the expression level of THPO. Results: The expression of THPO was increased in tumor tissue and cells, its high-regulation was associated with a poor prognosis in patients with gastric adenocarcinoma. Cell viability, invasion, and migration were suppressed in AGS with the down-regulation of THPO. Furthermore, on the basis of si-THPO transfection, E-cadherin was promoted while N-cadherin and Vimentin were attenuated. CONCLUSION Our results revealed that THPO may be a potent marker of gastric adenocarcinoma, providing a novel potential screening method for gastric adenocarcinoma.

RESUMO OBJETIVO Trombopoetina (THPO) é um conhecido fator de desenvolvimento e crescimento megacariócito (MGDF) envolvido na proliferação e maturação de megacariócitos. Realizamos este estudo para explorar os efeitos biológicos do THPO no adenocarcinoma gástrico. Metodologia: O nível de expressão do THPO em tecidos tumorais foi determinado acessando a banco de dados TCGA. A associação entre a expressão de THPO e características clínicas ou relevância no prognóstico foi descrita através da análise de Kaplan-Meier e regressão de Cox. O método SiRNA foi utilizado para reduzir a expressão da THPO e, em seguida, a viabilidade, invasão, e migração celular foram detectadas para verificar os efeitos da redução do THPO. qPCR e western blotting foram utilizados para examinar o nível de expressão do THPO. Resultados: A expressão do THPO estava aumentada em tecido e células tumorais, esse aumento estava associado com um prognóstico negativo para pacientes com adenocarcinoma gástrico. A invasão e migração celular foram suprimidos em AGS com a redução do THPO. Além disso, com base na transfecção de si-THPO, a E-caderina foi promovida, enquanto a N-caderina e Vimentina foram atenuadas. Conclusão nossos resultados demonstram que o thpo pode ser um potente marcador de adenocarcinoma gástrico, com potencial para ser um novo tipo de triagem para adenocarcinoma gástrico.

Anticancer drug screening: standardization of in vitro wound healing assay / Triagem de drogas anticâncer: padronização do ensaio de ranhura in vitro

ABSTRACT Introduction: Gliomas are characterized by rapid proliferation and aggressive invasion into normal surrounding brain tissue. In medical laboratories, the in vitro wound healing assay stands out as a simple, easy, inexpensive and affordable method to evaluate cell migration and proliferation. Objective: To standardize the in vitro wound healing assay using antimicrotubule drugs as positive controls. Methods: U87MG glioma cells were seeded at different densities and, after 24 h, the monolayer was scratched using different micropipette tip size to create a gap with no cells. The cells were then treated with colchicine and paclitaxel in culture medium with the presence or absence of fetal bovine serum. The wound was photographed with the aid of an inverted microscope and the wound area was measured using the Image J software. Results: Better defined edges scratches and monolayer with approximately 90% confluence were obtained at 1.5 and 2 x 105 cells/well density. The width and area of the scratch were, respectively, 948 pm/2.193221 mm2; 964 pm/2.266 mm2 and 1448 pm/3.221 mm2 to 10. 200 and 1000 pl micropipette tips. Colchicine inhibited wound closure by 12.6% or 3.4%, both in the presence or absence of serum; paclitaxel 2.4 and 6.7% respectively. Conclusion: Under standardized conditions, colchicine and paclitaxel proved to be efficient positive controls into the in vitro wound healing assay.

RESUMEN Introducción: Gliomas se caracterizan por rápida proliferación e invasion agresiva del tejido cerebral normal circundante. En laboratorios médicos, el ensayo de cierre de herida - una prueba in vitro - se destaca por ser un método simple, fácil, de bajo costo y accesiblepara evaluar la migración y la proliferación celular. Objetivo: Estandarizar el ensayo de cierre de herida usando agentes anti-microtúbulos como control positivo. Métodos: Las células de glioma U87MG fueron sembradas en diferentes concentracionesy, después de 24 horas, la monocamadafue rayada con punteras de diferentes tamanos para crear una hendidura sin células. Las células fueron entonces tratadas con colchicina y paclitaxel, en medio con o sin suero fetal bovino. La ranura fue fotografiada con la ayuda de un microscopio invertido, y el área de la ranura fue medida mediante el programa Image J. Resultados: Ranuras con bordes más bien-definidos y monocamada con alrededor de 90% de confluencia se obtuvieron con 1,5 y 2 x 105 células/pozo. La anchura y el área de las ranuras obtenidas fueron, respectivamente, 948 p.m/2,193 mm2; 964p.m/2,266mm2; y 1448p.m/3,221 mm2 para las punteras de 10,200y 1000pl. La colchicina inhibió el cierre de las ranuras en 12,6% o 2,4%, tanto en presencia como en ausencia de suero; el paclitaxel, 3,4% y 6,7%, respectivamente. Conclusión: En condiciones estandarizadas, colchicina y paclitaxel pueden ser usados como control positivo en el ensayo de cierre de herida in vitro.

RESUMO Introdução: Gliomas são caracterizados por terem rápida proliferação e invasão agressiva no tecido cerebral circundante normal. Em laboratórios médicos, o ensaio de ranhura - um teste in vitro - destaca-se por ser um método simples, fácil, barato e acessível para avaliar a migração e a proliferação celular. Objetivo: Padronizar o ensaio de ranhura, utilizando drogas antimicrotúbulos como controles positivos. Métodos: As células de glioma U87MG foram semeadas em diferentes concentrações e, após 24 horas, a monocamada foi arranhada usando ponteiras de diferentes tamanhos para criar uma fenda sem células. As células foram então tratadas com colchicina e paclitaxel, com meio em ausência ou presença de soro fetal bovino. A ranhura foi fotografada com auxílio de microscópio invertido, e a área da ranhura foi medida por meio do programa Image J. Resultados: Ranhuras com bordas mais bem definidas e monocamada com aproximadamente 90% de confluência foram obtidas com 1,5 e 2 x 105 células/poço. A largura e a área das ranhuras obtidasforam, respectivamente, 948pm/2,193 mm2;964pm/2,266mm2; e 1448 pm/3,221 mm2 para as ponteiras de 10, 200 e 1000 pl. A colchicina inibiu o fechamento das ranhuras em 12,6% ou 2,4%, tanto na presença quanto na ausência de soro; o paclitaxel, em 3,4% e 6,7%, respectivamente. Conclusão: Em condições padronizadas, colchicina e paclitaxel podem ser usados como controles positivos eficientes no teste in vitro de ranhura.

Determinación del efecto cicatrizante de Piper aduncum (matico) en fibroblastos humanos / Determination of the healing effect of Piper aduncum (spiked pepper or matico) on human fibroblasts

RESUMEN Objetivos. Evaluar el efecto cicatrizante del extracto hidroetanólico de Piper aduncum, en una línea celular de fibroblastos Dermales Adultos Humanos (hDFa). Materiales y métodos. El extracto se obtuvo mediante extracción sólido-líquido, fue concentrado y liofilizado. Se purificaron las proteínas del extracto mediante cromatografía líquida de alta eficacia de fase reversa (RP-HPLC); las proteínas fueron identificadas por espectrometría de masas en tándem de péptidos trípticos y se analizaron por MALDI-TOF-TOF en un espectrómetro de masa ABSciex4800. Los valores de concentración efectiva media (EC50), concentración inhibitoria media (IC50), y el porcentaje de proliferación celular; fueron determinados por ensayos con sales de tetrazolio (MTT) . La migración celular se evaluó mediante la "técnica de rayado" . Se analizó la expresión de factores de crecimiento mediante el ensayo de reacción en cadena de la polimerasa con transcriptasa reversa a tiempo real (RT- qPCR). Resultados. La línea hDFa evidenció un IC50 de 200 µg/mL con el extracto, el valor de EC50 fue 103,5 µg/mL. En el ensayo de proliferación, la proteína K2; mostró mayor actividad en la proliferación respecto de otros tratamientos (1 µg/mL). En el ensayo de migración de fibroblastos, la proteína K2 mostró mayor actividad (50 µg/mL). La expresión relativa del factor de crecimiento derivado de plaquetas (PDGF) se incrementó 8,6 veces respecto al control, en presencia de la proteína K2. Conclusiones. El extracto hidroetanólico, de Piper aduncum, así como las proteínas que contiene, incrementaron la proliferación y migración de fibroblastos dermales humanos (hDFa); así mismo, aumentaron la expresión de factores de crecimiento que intervienen en el proceso de cicatrización.

ABSTRACT Objectives. To evaluate the healing effect of a Piper aduncum ethanol-water extract on an adult human dermal fibroblast cell line (hDFa). Materials and Methods. After obtaining the extract via solid-liquid extraction, concentration, and lyophilization, extract proteins were purified using reverse phase high-performance liquid chromatography, identified using tandem mass spectrometry of tryptic peptides, and analyzed using MALDI-TOF-TOF on an ABSciex4800 mass spectrometer. Half maximum effective concentration values (EC50), half maximum inhibiting concentration (IC50), and percentages of cell proliferation were determined using tetrazolium salt assays. Cell migration was evaluated using a "scratch assay". Growth factor expression in cells was analyzed via quantitative real-time reverse transcription polymerase chain reaction. Results. Against the hDFa cell line, the extract had an IC50 of 200 μg/mL and EC50 of 103.5 µg/mL. In the proliferation assay, protein K2 (obtained from the extract) exhibited increased proliferative activity relative to other treatments (1 µg/mL); this agent also exhibited increased activity (50 µg/mL) in the fibroblast migration assay.Furthermore, the relative expression of platelet-derived growth factor increased by 8.6-fold in the presence of K2 protein relative to the control. Conclusions. The hydroethanolic extract of Piper aduncum and its component proteins increased the proliferation and migration of hDFa and increased the expression of growth factors involved in the healing process.

Expression and clinical significance of cortactin protein in ovarian neoplasms.

OBJECTIVE: To examine the expression of cortactin in epithelial ovarian cancer, and discuss the relationship between the expression of cortactin and the clinical pathology characteristics in epithelial ovarian cancer, as well as clinical significance. METHODS: The expression of cortactin was detected using real-time fluorescence quantitative PCR and immunohistochemical SP method in epithelial ovarian cancer. RESULTS: (1) The relative content of cortactin mRNA in epithelial ovarian cancer tissue was higher than that in benign control tissue, and expression was related to histological classification and FIGO stage. (2) Cortactin protein was localized in the cytoplasm and membrane of tumor cells. The positive rate of cortactin was 73.3 % in epithelial ovarian cancer, and the rate of cortactin expression was related to histological classification. (3) The average survival period of epithelial ovarian cancer patients with positive expression of cortactin was 19.5 ± 1.2 months (95 % CI 14.6-21.4 months), compared with 34.5 ± 4.3 months in the negative expression group (95 % CI 22.1-25.9 months). Univariate survival analysis showed that: negative expression of cortactin had a significant survival advantage (χ (2) = 5.739, P = 0.017). A cox regression model for multivariate analysis revealed that cortactin was an independent prognostic factor for epithelial ovarian cancer (P = 0.001; RR = 6.452, 95 % CI 2.289-7.112). CONCLUSIONS: Negative expression of cortactin was an independent prognostic factor and had a survival advantage. This suggested that cells with poor differentiation showed increasing motility. Cortactin is closely related to poor prognosis.

Effect of EDTA on TGF-ß1 released from the dentin matrix and its influence on dental pulp stem cell migration

Abstract: Bioactive molecules stored in dentin, such as transforming growth factor beta1 (TGF-b1), may be involved in the signaling events related to dental tissue repair. The authors conducted an in vitro evaluation of the amount of TGF-b1 released from dentin slices after treatment with 10% ethylenediaminetetraacetic acid (EDTA), 2.5% sodium hypochlorite (NaOCl) or phosphate-buffered saline (PBS), and the effect of this growth factor on stem cell migration from human exfoliated deciduous teeth (SHED). Sixty 1-mm-thick tooth slices were prepared with or without the predentin layer, and treated with either 10% EDTA for 1 minute, 2.5% NaOCl for 5 days or kept in PBS. Tooth slice conditioned media were prepared and used for TGF-b1 ELISA and migration assays. Culture medium with different concentrations of recombinant human TGF-b1 (0.5, 1.0, 5.0 or 10.0 ng/mL) was also tested by migration assay. The data were evaluated by ANOVA and Tukey's test. Optical density values corresponding to media conditioned by tooth slices either containing or not containing the predentin layer and treated with 10% EDTA were statistically greater than the other groups and close to 1 ng/mL. Increased rates of migration toward media conditioned by tooth slices containing the predentin layer and treated with PBS, 10% EDTA or 2.5% NaOCl were observed. Recombinant human TGF-b1 also stimulated migration of SHED, irrespective of the concentration used. EDTA may be considered an effective extractant of TGF-b1 from the dentin matrix. However, it does not impact SHED migration, suggesting that other components may account for the cell migration.

Participação das proteínas moesina e Rho-A na evolução dos tumores odontogênicos benignos / Participation of moesin and Rho-A proteins in evolution of benign odontogenic tumors

A moesina, uma das proteínas do complexo ERM (ezrina, radixina e moesina), está envolvida nos processos de migração e invasão tumoral, participando da dinâmica do citoesqueleto na movimentação celular associada à ativação da GTPase Rho-A. O objetivo desse estudo foi avaliar a correlação da imunoexpressão da moesina e da Rho-A em tumores odontogênicos benignos, diagnosticados no Serviço de Anatomia Patológica da Faculdade de Odontologia de Bauru (USP), no período de 1963 a 2009. Um total de 45 tumores odontogênicos benignos incluindo 7 ameloblastomas, 8 tumores odontogênicos adenomatóides, 19 tumores odontogênicos queratocísticos, 2 cistos odontogênicos ortoqueratinizantes, 1 tumor odontogênico epitelial calcificante, 2 fibromas ameloblásticos, 4 fiboodontomas ameloblásticos e 2 tumores odontogênicos císticos calcificantes, foram avaliados quanto a expressão imunohistoquímica da moesina e da Rho-A pelas células odontogênicas. A correlação entre as expressões membranosa e citoplasmática da moesina e da Rho-A pelo epitélio odontogênico nos diferentes tumores foi avaliada pelo teste de correlação de Spearman, com nível de significância de 5%. Os resultados mostraram uma forte expressão membranosa de moesina e citoplasmática de Rho-A em 66,7% e 62,2% dos tumores odontogênicos benignos, respectivamente. Houve uma correlação positiva e estatisticamente significativa entre a expressão membranosa e citoplasmática da moesina (ρ=0,000) e de Rho-A (ρ=0,048) nos tumores. Entretanto, não houve correlação entre as expressões demoesina e de Rho-A nos tumores odontogênicos benignos. Estes resultados comprovam que a moesina e a Rho-A são fortemente expressas pelo epitélio odontogênico neoplásico e, sugerem que ambas proteínas provavelmente participamdo crescimento e expansão local destes tumores odontogênicos benignos.

The moesin, one of the proteins of the ERM complex (ezrin, radixin and moesin), is involved in the migration and tumor invasion processes participating in the cytoskeleton dynamics in cell movement associated with the activation of the GTPase Rho-A. The aim of this study was to evaluate the immunoexpression orrelation of moesin and Rho-A in benign odontogenic tumors, diagnosed at the Bauru School of Dentistry Oral Pathology Biopsy Service of the University of São Paulo in the period of 1963-2009. A total of 45 benign odontogenic tumors including 7 ameloblastomas, 8 adenomatoid odontogenic tumors, 19 keratocystic odontogenic tumors, 2 orthokeratinized odontogenic cyst, 1 calcifying epithelial odontogenic tumor, 2 ameloblastic fibroma, 4 ameloblastic fibroodontoma and 2 calcifying cystic odontogenic tumors, were evaluated for immunohistochemical expression of moesin and Rho-A by odontogenic cells. The correlation between the membranous and cytoplasmic expression of moesin and Rho-A by the odontogenic epithelium in different tumors was evaluated by the Spearman correlation test, with a significance level of 5%. The results showed strong membranous expression of moesin and cytoplasmic expression of Rho-A in 66.7% and 62.2% of the benign odontogenic tumors, respectively. There was a positive and statistically significant correlation between membranous and cytoplasmic expression of moesin (ρ=0.000) and Rho-A (ρ=0.048) in the tumors. However, there was no correlation between the expression of moesin and Rho-A in benign odontogenic tumors. These results show that the moesin and Rho-A are strongly expressed by neoplastic odontogenic epithelium and suggest that both proteins probably participate in the growth and local expansion of these benign odontogenic tumors.

The Tomato spotted wilt virus cell-to-cell movement protein (NSM ) triggers a hypersensitive response in Sw-5-containing resistant tomato lines and in Nicotiana benthamiana transformed with the functional Sw-5b resistance gene copy.

Although the Sw-5 gene cluster has been cloned, and Sw-5b has been identified as the functional gene copy that confers resistance to Tomato spotted wilt virus (TSWV), its avirulence (Avr) determinant has not been identified to date. Nicotiana tabacum 'SR1' plants transformed with a copy of the Sw-5b gene are immune without producing a clear visual response on challenge with TSWV, whereas it is shown here that N. benthamiana transformed with Sw-5b gives a rapid and conspicuous hypersensitive response (HR). Using these plants, from all structural and non-structural TSWV proteins tested, the TSWV cell-to-cell movement protein (NSM ) was confirmed as the Avr determinant using a Potato virus X (PVX) replicon or a non-replicative pEAQ-HT expression vector system. HR was induced in Sw-5b-transgenic N. benthamiana as well as in resistant near-isogenic tomato lines after agroinfiltration with a functional cell-to-cell movement protein (NSM ) from a resistance-inducing (RI) TSWV strain (BR-01), but not with NSM from a Sw-5 resistance-breaking (RB) strain (GRAU). This is the first biological demonstration that Sw-5-mediated resistance is triggered by the TSWV NSM cell-to-cell movement protein.

Cyclosporin safety in a simplified rat brain tumor implantation model / Segurança do tratamento com ciclosporina em um modelo pré-clínico de tumor cerebral experimental em ratos

Brain cancer is the second neurological cause of death. A simplified animal brain tumor model using W256 (carcinoma 256, Walker) cell line was developed to permit the testing of novel treatment modalities. Wistar rats had a cell tumor solution inoculated stereotactically in the basal ganglia (right subfrontal caudate). This model yielded tumor growth in 95 percent of the animals, and showed absence of extracranial metastasis and systemic infection. Survival median was 10 days. Estimated tumor volume was 17.08±6.7 mm³ on the 7th day and 67.25±19.8 mm³ on 9th day post-inoculation. Doubling time was 24.25 h. Tumor growth induced cachexia, but no hematological or biochemical alterations. This model behaved as an undifferentiated tumor and can be promising for studying tumor cell migration in the central nervous system. Dexamethasone 3.0 mg/kg/day diminished significantly survival in this model. Cyclosporine 10 mg/kg/day administration was safely tolerated.

Neoplasias encefálicas constituem a segunda causa neurológica de morte. Foi desenvolvido um modelo animal simplificado de tumor cerebral em ratos utilizando a linhagem celular W256 (carcinoma 256 de Walker) para permitir teste de novos tratamentos. Ratos Wistar foram inoculados nos gânglios da base (caudato subfrontal direito) com uma solução celular tumoral, por via estereotáxica. Este modelo demonstrou crescimento tumoral em 95 por cento dos animais inoculados com sucesso, além de mostrar ausência de metástases extracranianas e infecção sistêmica. A mediana de sobrevida dos animais foi de 10 dias. O volume tumoral estimado foi de 17,08±6,7 mm³ no sétimo dia e de 67,25±19,8 mm³ no nono dia após a inoculação. O tempo de duplicação foi estimado em 24,25 h. O crescimento tumoral induziu a caquexia, mas não houve alterações bioquímicas ou hematológicas. Esse modelo permite fácil reprodução e comporta-se como um tumor indiferenciado, mostrando potencial para estudar migração celular tumoral no sistema nervoso central. Dexametasona 3,0 mg/kg/dia reduziu significantemente a sobrevida dos animais inoculados com tumor nesse modelo. Ciclosporina 10 mg/kg/dia não teve efeito na sobrevida, sendo sua administração bem tolerada.