RESUMEN
This study is designed to screen the CD40 related signal transduction pathway in AGS cells and construction of gene silencing vector. Analysis results showed 414 differential genes expression, including upregulation of 209 genes and downregulation of 205 genes. Basing on the ratio of signal in experimental group to signal in control group, 45 genes (38 genes upregulation and seven genes downregulation) with significant (P<0.01) change in expression levels were screened according to the screening standard (signal log ratio≥1 or ≤-1). These genes involved into metabolism, cell cycle and apoptosis, signal transduction and stress response. Furthermore, PI3K mRNA expression level in PI3K siRNA transfected AGS cells was 0.2335±0.0116 72 h after transfection. This value was significantly (P<0.05) lower than that in blank and negative control groups. PI3K protein expression in PI3K siRNA transfected AGS cells was significantly (P<0.05) lower than that in blank and PI3K siRNA/N transfected groups. Therefore, PI3K siRNA gene silencing vector can significantly inhibit PI3K mRNA and protein expression in AGS cells.
Asunto(s)
Antígenos CD40/metabolismo , Regulación de la Expresión Génica/genética , Vectores Genéticos/genética , Transducción de Señal/genética , Análisis de Varianza , Western Blotting , Línea Celular Tumoral , Biología Computacional , Cartilla de ADN/genética , Perfilación de la Expresión Génica , Silenciador del Gen , Humanos , Microscopía Fluorescente , Fosfatidilinositol 3-Quinasas/metabolismo , ARN Interferente Pequeño/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección/métodosRESUMEN
In this study, we investigate effect of PI3K gene silencing on growth, migration and related proteins expression of CD40 signal-mediated gastric cancer cells. We observed that combination of sCD40L with PI3K siRNA could significantly inhibit AGS cells growth, block cells in G1 phase, and promote tumour cells apoptosis after 24 h treatment. Transwell test showed that numbers of cells per visual field in group PI3K siRNA or group sCD40L (after 24 h PI3K siRNA or sCD40L alone treatment) were fewer than that (32.54 ± 4.22) in control group. Numbers of cells per visual field in (after 24 h combination treatment of PI3K siRNA with sCD40L) were significantly fewer than that in group PI3K siRNA or group sCD40L. Compared with group sCD40L, expression level of Fas protein in group sCD40L + PI3K siRNA was significantly increased. The findings suggest that PI3K siRNA may strengthen CD40-induced specific antitumour effect via blocking PI3K/Akt signal pathway, resisting tumour immunoediting regulated by CD40 signal. Combination of sCD40L and PI3K siRNA is an important mechanism of gastric cancer therapy.
Asunto(s)
Ligando de CD40/genética , Movimiento Celular , Proliferación Celular , Fosfatidilinositol 3-Quinasas/genética , Interferencia de ARN , Apoptosis , Antígenos CD40/metabolismo , Ligando de CD40/metabolismo , Ciclo Celular , Línea Celular Tumoral , Forma de la Célula , Supervivencia Celular , Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Proteínas Inhibidoras de la Apoptosis/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Survivin , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor fas/metabolismoRESUMEN
The aim of this study was to investigate the molecular mechanisms underlying the antitumour effects of CD40L through analysing the change of genes expression profile in AGS using Affymetrix Gene Chip. Human gastric carcinoma AGS cells were first incubated with 2 µg/ml sCD40L or equal volume of medium (control) in F12 medium. RNA was isolated from AGS and were reverse transcribed, labeled with digoxigenin-11-dUTP, and then hybridized with Clontech Atlas mouse cDNA expression arrays for comparison. Performing clustering analysis, we found that 7 detected genes were down-regulated and 38 were upregulated as the sCD40L acted on AGS. To further verify the results of gene chip screening, Gene Database was searched, finding that the most significantly up-regulated genes were Gadd45a, c-Jun and Bcl-2, and the most significantly down-regulated genes were Cyclin D1, CDC6, TNFR10B, c-IAP2 and ORC5L. Based upon these findings, the signalling pathways that possibly mediate CD40-induced apoptosis are proposed.
Asunto(s)
Antineoplásicos/farmacología , Antígenos CD40/metabolismo , Ligando de CD40/farmacología , Expresión Génica/efectos de los fármacos , Transducción de Señal , Línea Celular Tumoral , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Neoplasias Gástricas , Temperatura de TransiciónRESUMEN
CD40 signaling plays a critical role in the survival rate of gastric cancer patients. Tumour samples were collected from 73 patients with who were diagnosed as gastric cancer in general surgery department in the 1st affiliated hospital of Suzhou University between September 2002 and July 2003. All patients had not received radiotherapy and chemotherapy before operation. These patients include 46 male and 27 female. Here we show that CD40 is constitutively expressed in the human gastric carcinoma tissues, and CD40 protein and mRNA positive expression in gastric cancer tissues closely correlated with lymph node metastasis and tumour TNM stage. CD40 positive expression in gastric cancer patients with lymph node metastasis was markedly higher than that in gastric cancer patients without lymph node metastasis. CD40 positive expression in stage III-IV gastric cancer patients was markedly higher than that in stage I-II gastric cancer patients. Moreover, CD40 expression closely correlated with prognosis of gastric cancer patients. Therefore, CD40 was taken as grouping variable, and lymph node metastasis and clinical staging were taken as stratification variables, respectively, further analysis showed that prognosis in gastric cancer patients with lymph node metastasis and CD40 positive expression was markedly worse than that in gastric cancer patients without lymph node metastasis and CD40 negative expression (P = 0.0076). These results suggest that CD40 signaling plays a critical role in the survival of gastric cancer patients.
Asunto(s)
Biomarcadores de Tumor/genética , Antígenos CD40/genética , Neoplasias Gástricas/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/metabolismo , Antígenos CD40/metabolismo , Femenino , Humanos , Ganglios Linfáticos/metabolismo , Ganglios Linfáticos/patología , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pronóstico , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidadRESUMEN
The objective of the paper is to investigate the combined effect of sCD40L and phosphatidylinositol 3-kinase (PI3K) siRNA on transplanted tumours growth and microenvironment in nude mice with gastric cancer. 48 h after modeling, the animals were randomly divided into saline + AGS group (A), sCD40L + AGS group (B), saline + PI3K siRNA group (C) and sCD40L + PI3K siRNA group (D), six mice in each group. The mouse in the groups was inoculated with exponential phase AGS cell or PI3K gene silencing cells (100 µl, 5 × 10(6)). After tumour size reaches 0.2-0.3 cm, Tumours in animals of groups were injected with sCD40L (100 µl, 10 mg/kg) or equal volume of saline, thrice each day, respectively. Microvessel density (MVD), apoptosis index, and expression levels of PI3K, Survivin and vascular endothelial growth factor (VEGF) proteins in transplanted tumor cells in gastric cancer nude mice were analyzed by utilizing Immunohistochemistry, western blot, terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Results showed that combination of sCD40L with PI3K siRNA could significantly decrease tumour size, MVD, expression levels of PI3K, Survivin and VEGF proteins, and increase apoptosis index. It can be concluded that combination of sCD40L with PI3K siRNA provides a promising future for gastric cancer therapy.
Asunto(s)
Fosfatidilinositol 3-Quinasa/genética , Interferencia de ARN , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Microambiente Tumoral , Animales , Apoptosis/genética , Ligando de CD40 , Femenino , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Ratones , Ratones Desnudos , Neovascularización Patológica , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Neoplasias Gástricas/patología , Survivin , Carga Tumoral , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Two GTPases in the signal recognition particle (SRP) and SRP receptor (SR) interact with one another to mediate the cotranslational protein targeting pathway. Previous studies have shown that a universally conserved SRP RNA facilitates an efficient SRP-SR interaction in the presence of a signal sequence bound to SRP. However, a remarkable exception has been found in chloroplast SRP (cpSRP) pathway, in which the SRP RNA is missing. Based on biochemical and structural analyses, it is proposed that free cpSRP receptor (cpFtsY) has already been preorganized into a closed state for efficient cpSRP-cpFtsY association. However, no direct evidence has been reported to support this postulation thus far. In this study, we characterized the structural dynamics of cpFtsY and its conformational rearrangements induced by GTP binding using molecular dynamics (MD) simulations. Our results showed that the GTP-binding event triggered substantial conformational changes in free cpFtsY, including the relative orientation of N-G domain and several conserved motifs that are critical in complex formation. These rearrangements enabled the cpFtsY to relax into a preorganized 'closed' state that favored the formation of a stable complex with cpSRP54. Interestingly, the intrinsic flexibility of αN1 helix facilitated these rearrangements. In addition, GTP binding in cpFtsY was mediated by conserved residues that have been shown in other SRP GTPases. These findings suggested that GTP-bound cpFtsY could fluctuate into conformations that are favorable to form the stable complex, providing explanation of why SRP-SR interaction bypasses the requirement of the SRP RNA at a molecular level.
Asunto(s)
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Conformación Proteica , Proteínas de Cloroplastos , Partícula de Reconocimiento de Señal/metabolismoRESUMEN
The CD40 signaling pathway plays a key role in tumor cell proliferation, differentiation, and apoptosis. Gastric cancer usually possesses a higher level of CD40 expression than normal tissue. We evaluated inhibition of soluble CD40 ligand (sCD40L) in apoptosis induction of gastric cancer cells. Gastric cancer cells (AGS and BGC-823) were incubated with sCD40L. Cell viability and cell cycle were determined by methyl-tetrazolium (MTT) assay and flow cytometry, respectively. The results showed that sCD40L hindered cell growth, arrested cells at G0/G1 phase and induced apoptosis. In conclusion, sCD40L suppress growth of gastric cancer cells through apoptosis induction and cell cycle quiescence. This work will provided a new approach to gene therapy of gastric cancer.
Asunto(s)
Ligando de CD40/farmacología , Neoplasias Gástricas/patología , Apoptosis/efectos de los fármacos , Bioensayo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Solubilidad/efectos de los fármacosRESUMEN
To study expression of CD40 and CD40L in gastric cancer tissue we assessed gastric cancer patients admitted to the Department of Gastroenterology of The First Affiliated Hospital of Soochow University and control subjects. Gastric cancer and normal (from around tumours) tissue samples were obtained from patients. Venous blood samples (gastric cancer and ulcer groups) were drawn on the morning of the day before surgery for the measurement of peripheral sCD40L. The expression of CD40 in gastric carcinoma specimens was examined immuno-histochemically. The clinicopathological factors, including age, sex, tumor size, gross appearance, degree of cellular differentiation, histological classification, depth of tumor invasion, lymph node metastasis, peritoneal dissemination, and TNM stage were analyzed according to the different expression of CD40. The results indicated a high CD40 expression in gastric cancer tissues. This positive expression of CD40 revealed a significant (P < 0.05) correlation with lymphatic metastasis and tumor TNM stage in gastric cancer patients. It is concluded that higher CD40 expression existed in expanding type tumors and could play an important role in clinical diagnosis of gastric cancer patients.
Asunto(s)
Antígenos CD40/metabolismo , Ligando de CD40/sangre , Neoplasias Gástricas/metabolismo , Adulto , Anciano , Estudios de Casos y Controles , Femenino , Gelsolina/metabolismo , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Gástricas/patología , Neoplasias Gástricas/cirugía , Sindecano-1/metabolismo , Factor de Transcripción ReIA/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adulto JovenRESUMEN
UNLABELLED: OBJECTIVE; To investigate the expression of CD40 in gastric cancer and explore the correlation of CD40 with the outcome of patients. METHODS: The expression of CD40 was examined by immunohistochemistry in human gastric cancer tissues (73 cases) and adjacent normal gastric tissues (51 cases). CD40 mRNA was detected by RT-PCR in 11 gastric cancer and 11 adjacent normal gastric tissues. The patients were followed up for 60 months. The correlation of CD40 expression and the overall survival time were evaluated by Kaplan-Meier chart and Log-Rank test. Cox model was used for multivariate analysis. RESULTS: The immunoreactivity of CD40 increased in 35/73 gastric cancer tissues (47.9%) as compared to the tumor-free tissues (P = 0.0063). CD40 mRNA were detected in 72.7% (8/11) gastric cancer tissues, while not in all tumor-free tissues. After a 5-year follow-up, the survival rate was 57.9% (22/38) in CD40-negative patients, and 5.7% (2/35) in CD40-positive patients (P = 0.0022). The median survival time of patients with negative, positive, and strong CD40 expression was 56, 29 and 11 months respectively (P = 0.0085). Cox regression analysis suggested that CD40, lymph node metastasis and TNM stage were independent factors affecting the prognosis of gastric cancer patients. CONCLUSION: Normal and malignant gastric tissues are found to have its own unique CD40 expression patterns. CD40 may play a role in metastatic spread of gastric cancer and its expression may be used as an important survival predictor in human gastric cancer.