RESUMEN
Emerging evidence has linked aberrantly expressed microRNAs (miRNAs) with oncogenesis and malignant development in various human cancers. However, their specific roles and functions in gastric carcinoma (GC) remain largely undefined. In this study we identify and report a novel miRNA, miR-1225-5p, as tumor suppressor in GC development and progression. Microarray analysis revealed that there were fifty-six differentially expressed miRNAs (thirty-two upregulated and twenty-four downregulated) in GC tumor samples compared to their corresponding nontumorous tissues. Downregulation of miR-1225-5p was frequently detected in GC and strongly correlated with more aggressive phenotypes and poor prognosis. Functional assays demonstrated that ectopic overexpression of miR-1225-5p could inhibit cell proliferation, colony formation, migration and invasion in vitro, as well as suppress tumor growth and metastasis in nude mice. Further integrative and functional studies suggested insulin receptor substrate 1 (IRS1) as a downstream effector of miR-1225-5p which acted through ß-catenin signaling pathway. These results demonstrate that miR-1225-5p serves to constrain GC growth and metastatic potential via inhibition of IRS1 and ß-catenin signaling. Therefore, downregulation of miR-1225-5p is likely to be one of major molecular mechanisms accounting for the development and progression of GC.
Asunto(s)
Adenocarcinoma/secundario , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Proteínas Sustrato del Receptor de Insulina/metabolismo , MicroARNs/genética , Neoplasias Gástricas/patología , beta Catenina/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Apoptosis , Western Blotting , Movimiento Celular , Femenino , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Humanos , Técnicas para Inmunoenzimas , Proteínas Sustrato del Receptor de Insulina/genética , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Persona de Mediana Edad , Invasividad Neoplásica , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genéticaRESUMEN
Thrombospondin 1 (THBS1) plays an important role in angiogenesis and tumor progression. The aim of the present study was to investigate the effects of single-nucleotide polymorphisms (rs1478605 and rs3743125) in the untranslated regions of the THBS1 gene on the development and progression of gastric cancer. In the case-control study, 275 gastric cancer patients and 275 cancer-free controls were successfully genotyped using polymerase chain reaction-restriction fragment length polymorphism. The data demonstrated that THBS1 rs1478605 genotypic distributions significantly differed between the patient and control groups (P=0.005). Carriers of the CC genotype exhibited a decreased risk of developing gastric cancer compared to the carriers of the CT and TT genotypes [adjusted odd ratio (OR), 0.56; 95% confidence interval (CI), 0.39-0.79; P=0.001]. The CC genotype of rs1478605 was negatively associated with gastric cancer lymph node metastasis (OR, 0.41; 95% CI, 0.23-0.71; P=0.001) and was associated with a reduced risk of lymph node metastasis in male patients (OR, 0.27; 95% CI, 0.14-0.52; P<0.001). The THBS1 CT haplotype was associated with a reduced risk of developing gastric cancer (OR, 0.56; 95% CI, 0.33-0.93; P=0.02). By contrast, no association was observed between THBS1 rs3743125 and the development and progression of gastric cancer. These results suggest that THBS1 rs1478605 represents a potential molecular marker for gastric cancer.
RESUMEN
As a member of the Myc proto-oncogene family, MYCL1 has been found to be amplified and overexpressed in some malignancies. However, the clinical significance of Mycl1 expression in gastric cancer is still unknown. Mycl1 expression was detected on tissue microarrays of gastric cancer samples in 176 cases using immunohistochemical staining, and its association with clinicopathological factors and overall survival was also analyzed. Mycl1 showed greater expression in gastric cancer tissue than in adjacent normal tissue (62.5% vs 46.0%, respectively, P=0.002), and its expression was correlated with patient age, tumor differentiation, and TNM stage (P=0.007, 0.003, and 0.002, respectively). The Mycl1 positive group had an unfavorable outcome compared with the negative group (P<0.001). Multivariate analysis showed that Mycl1 expression was an independent prognostic factor of gastric cancer (P=0.009). These results suggest that Mycl1 expression might be useful as a biomarker to predict prognosis and is a promising therapeutic target for patients with gastric cancer.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc/metabolismo , Neoplasias Gástricas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Mucosa Gástrica/metabolismo , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Proto-Oncogenes Mas , Estómago/patología , Neoplasias Gástricas/patologíaRESUMEN
Previous studies have shown that transfection of the snake venom cystatin (sv-cystatin) gene can inhibit the invasion and metastasis of tumor cells. The aim of this study was to investigate the pharmaceutical applications of sv-cystatin in melanoma gene therapy. We constructed a recombinant adenovirus carrying sv-cystatin (Ad/sv-cystatin) and a control virus (Ad/null). Matrigel assays were used to assess melanoma cell migration and invasiveness in vitro. The antimelanoma effects of Ad/sv-cystatin were assessed in a syngeneic mouse model with an experimental lung colonization assay. Ad/sv-cystatin significantly inhibited the invasion and growth of B16F10 cells in vitro compared with control and Ad/null. Ad/sv-cystatin significantly inhibited experimental lung colonization in C57BL/6 mice as compared with that in control (P<0.001) and Ad/null-treated mice (P<0.001), with an inhibition rate of 51 and 46%, respectively. Ad/sv-cystatin slowed the increase in lung weight in C57BL/6 mice as compared with that in control mice (P<0.001) and Ad/null-treated mice (P<0.001), with an inhibition rate of 40 and 35%, respectively. Our results indicate that Ad/sv-cystatin suppresses mouse melanoma invasion, metastasis, and growth in vitro and in vivo. Our findings provide support for the further examination of the pharmaceutical applications of Ad/sv-cystatin.
Asunto(s)
Cistatinas/farmacología , Melanoma Experimental/tratamiento farmacológico , Melanoma Experimental/patología , Venenos de Serpiente/química , Adenoviridae/metabolismo , Animales , Movimiento Celular , Proliferación Celular/efectos de los fármacos , Colágeno , Combinación de Medicamentos , Terapia Genética/métodos , Laminina , Masculino , Ratones , Ratones Endogámicos C57BL , Invasividad Neoplásica , Metástasis de la Neoplasia/tratamiento farmacológico , Trasplante de Neoplasias , Proteoglicanos , Proteínas Recombinantes/farmacología , TransfecciónRESUMEN
Studies have shown that the recombinant BJ46a (rBJ46a) protein can reduce matrix metalloproteinase (MMP) activities and inhibit invasion and metastasis of melanoma cells. Here, we optimized the Pichia pastoris system to evaluate rBJ46a protein as an anticancer agent. The Enzchek gelatinase/collagenase assay showed that rBJ46a inhibited MMP activities (IC50=0.119 mg/ml). Kinetic analyses using a series of double reciprocal Lineweaver-Burk plots (1/V vs. 1/S) showed a competitive mode of inhibition with rBJ46a with inhibitory efficiency against MMPs (Ki=13.6 nmol/l). Matrigel invasion assays showed significant activity of rBJ46a on tumor cells. For lung colonization assays, C57BL/6 mice were inoculated in the lateral tail vein with B16F10 cells and were treated with three i.v. injections of rBJ46a (1, 2, and 4 mg/kg) 24 h before cell inoculation, and 2 and 24 h after cell inoculation. Administration of rBJ46a suppressed lung tumor colony formation significantly. For spontaneous metastasis assays, MHCC97H cells were inoculated subcutaneously into nude mice. After 24 h, rBJ46a was administered by i.p. injections: 1, 2, and 4 mg/kg once daily for 6 days. rBJ46a decreased lung tumor colony formation significantly. Gelatin zymography showed that MMP2/MMP9 enzymatic activities in tumor cells were suppressed by rBJ46a in a dose-dependent manner, and the Km values of rBJ46a against MMP2 and MMP9 activities that were expressed in both B16F10 and MHCC97H cells were 3.6 and 1.4 µmol/l, respectively. Thus, rBJ46a can inhibit the invasion and metastasis of tumor cells by reducing MMP2/MMP9 activities, indicating that rBJ46a may be a novel therapeutic agent for antimetastasis of tumor cells.
Asunto(s)
Antineoplásicos/farmacología , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/farmacología , Venenos de Víboras/farmacología , Animales , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales/métodos , Humanos , Concentración 50 Inhibidora , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Melanoma Experimental/tratamiento farmacológico , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Pichia/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Venenos de Víboras/genética , Venenos de Víboras/aislamiento & purificaciónRESUMEN
To explore the association of polymorphisms in the region of three neighboring genes TRIT1, MYCL1 and MFSD2A with risk and clinicopathological features of gastric cancer, 19 tagging SNPs in this region were genotyped using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in a case-control study of 610 Chinese gastric cancer patients and 608 cancer-free controls. MFSD2A rs4233508 T>C CC genotype was associated with an increased risk of gastric cancer in younger patients and an increased risk of moderately/well-differentiated intestinal-type gastric cancer (adjusted odds ratio [OR], 1.74 and 1.50, respectively). TRIT1 rs11581557 T>G TG was associated with lymph node metastasis (TG versus TT/GG, adjusted OR, 1.64). MFSD2A rs12083239 GC genotype and TRIT1 rs2172362 or rs230310 homozygous genotype were associated with Lauren's classification (GC versus GG, adjusted OR, 1.69; GC versus GG/CC, adjusted OR, 1.74) and tumor site (rs2172362: CC versus CT, adjusted OR, 1.71; CC/TT versus CT, adjusted OR, 1.62; rs230310: CC versus CT, adjusted OR, 1.75; CC/TT versus CT, adjusted OR, 1.67) of gastric cancer, respectively. One TRIT1 haplotype, CCGT, was associated with lymph node metastasis and tumor site of gastric cancer (CCGT versus TTTT, adjusted OR, 1.91 and 1.55). This is believed to be the first report that several tagging SNPs and haplotypes in TRIT1, MYCL1 and MFSD2A region are significantly associated with risk and clinicopathological features of gastric cancer in a Chinese population. The findings might be useful for risk assessment and prognosis prediction of gastric cancer.
Asunto(s)
Transferasas Alquil y Aril/genética , Genes myc , Neoplasias Gástricas/genética , Proteínas Supresoras de Tumor/genética , Pueblo Asiatico , Estudios de Casos y Controles , Femenino , Predisposición Genética a la Enfermedad , Haplotipos , Humanos , Metástasis Linfática/genética , Masculino , Polimorfismo de Nucleótido Simple , Neoplasias Gástricas/enzimología , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , SimportadoresRESUMEN
Previous studies have shown that recombinant snake venom cystatin (sv-cystatin) inhibits the invasion and metastasis of tumor cells in vitro and in vivo. The purpose of this study was to investigate the ability of recombinant sv-cystatin to inhibit tumor angiogenesis in vitro and in vivo, and the mechanisms underlying this effect. Recombinant sv-cystatin inhibited proliferation of human umbilical vein endothelial cells (HUVECs) at 100 and 200 µg/mL after 72, 96 and 120 h. Recombinant sv-cystatin also inhibited tumor-endothelial cell adhesion at 25, 50, 100 and 200 µg/mL. Recombinant sv-cystatin inhibited capillary-like tube formation by HUVECs at 10, 25, 50, 100 and 200 µg/mL following 12, 24 and 36 h incubation. Furthermore, recombinant sv-cystatin significantly suppressed microvessel density (MVD) of lung tumor colonies in C57BL/6 mice inoculated in the lateral tail vein with B16F10 melanoma cells. Administration of recombinant sv-cystatin significantly decreased MVD of primary tumor tissues in nude mice implanted subcutaneously with human hepatocellular carcinoma cells (MHCC97H). Exposure of B16F10 and MHCC97H cells to increasing doses of recombinant sv-cystatin suppressed secretion of vascular endothelial growth factor (VEGF)-A165 and basic fibroblast growth factor (bFGF) into the surrounding medium (P < 0.05). The expression of fms-related tyrosine kinase 1 (Flt-1) protein in HUVECs was decreased by 25, 50, 100 and 200 µg/mL recombinant sv-cystatin (P < 0.05). This study demonstrates that recombinant sv-cystatin inhibits tumor angiogenesis associated with downregulation of VEGF-A165, Flt-1 and bFGF. This suggests that recombinant sv-cystatin may have potential pharmaceutical applications as an antiangiogenic and antimetastatic therapeutic agent.
Asunto(s)
Cistatinas/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Neoplasias Experimentales/metabolismo , Neovascularización Patológica/metabolismo , Venenos de Serpiente/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Proliferación Celular , Cistatinas/química , Cistatinas/aislamiento & purificación , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Células Endoteliales de la Vena Umbilical Humana/citología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Desnudos , Proteínas Recombinantes , Venenos de Serpiente/química , Venenos de Serpiente/aislamiento & purificación , Relación Estructura-Actividad , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
A multicomponent DNA vaccine, encoding Toxoplasma gondii GRA1 and SAG1, was constructed and tested for its ability to confer protection. BALB/c mice were challenged with tachyzoites of the virulent T. gondii RH strain at 4 weeks following the last immunization, and immune responses and survival times were observed. The results show that vaccination by the multicomponent vaccine prolonged survival of mice challenged with the T. gondii RH strain (from average 4.50 ± 0.22 to 7.60 ± 0.74 days); induced high levels of IgG antibody (from 0.252 ± 0.080 to 0.790 ± 0.083), IFN-gamma (from 598.74 ± 67.50 to 853.77 ± 66.74 pg/ml), and IL-2 (from 89.44 ± 10.66 to 192.24 ± 19.90 pg/ml); changed the CD4(+)/CD8(+) lymphocyte ratio (from 1.81 ± 0.14 to 1.09 ± 0.19); and stimulated NK cell-killing activity (from 46.81 ± 3.96 to 64.15 ± 7.71 %). These findings demonstrate that a multicomponent DNA vaccine, encoding GRA1 and SAG1, primes a strong humoral and cellular immune response and enhances protection against T. gondii challenge. The new, combined DNA vaccine provides another means to combat T. gondii infection.
Asunto(s)
Antígenos de Protozoos/inmunología , Proteínas Protozoarias/inmunología , Toxoplasma/inmunología , Vacunas de ADN/inmunología , Animales , Anticuerpos Antiprotozoarios/sangre , Antígenos de Protozoos/genética , Relación CD4-CD8 , Modelos Animales de Enfermedad , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Interleucina-2/metabolismo , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos BALB C , Proteínas Protozoarias/genética , Análisis de Supervivencia , Toxoplasma/genética , Toxoplasmosis Animal/inmunología , Toxoplasmosis Animal/prevención & control , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genéticaRESUMEN
Snake venom metalloproteinase inhibitor BJ46a is from the serum of the venomous snake Bothrops jararaca. It has been proven to possess the capacity to inhibit matrix metalloproteinases (MMPs), likely based on its structural similarity to MMPs. This report describes the successful expression, purification, and characterization of the recombinant protein BJ46a in Pichia pastoris. Purified recombinant protein BJ46a was found to inhibit MMPs. Structural modeling was completed and should provide the foundation for further functional research. To our knowledge, this is the first report on the large scale expression of BJ46a, and it provides promise as a method for generation of BJ46a and investigation of its potential use as a new drug for treatment of antitumor invasion and metastasis.
Asunto(s)
Metaloendopeptidasas/antagonistas & inhibidores , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Venenos de Víboras/biosíntesis , Venenos de Víboras/aislamiento & purificación , Secuencia de Aminoácidos , Clonación Molecular , Biología Computacional , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Pichia/química , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Venenos de Víboras/química , Venenos de Víboras/genéticaRESUMEN
BACKGROUND AND OBJECTIVES: The roles of thrombospondin-1 (THBS-1) in tumor growth and metastasis are complicated and its function as a cancer inhibitor or promoter remains controversial. This clinical study investigated the functional roles of THBS-1 in gastric carcinoma by examining the expression patterns of THBS-1 protein and mRNA levels during gastric cancer development. METHODS: Eighty-two gastric carcinomas were included in this study. THBS-1, α-smooth muscle actin, and CD34 proteins were localized by immunohistochemical staining, and the levels of THBS-1 mRNA were quantified by real-time polymerase chain reaction. RESULTS: THBS-1 mRNA expression in gastric carcinoma tissues was significantly higher than in adjacent non-cancerous stomach tissues (P = 0.03). Tumor THBS-1 mRNA expression level was significantly related to lymph node metastasis (P = 0.031), tumor size (P = 0.021) and patient age (P = 0.005). THBS-1 protein was mainly located in stromal myofibroblasts, and was undetectable in tumor cells. Myofibroblasts may be mainly derived from stromal fibroblasts in gastric cancer. The abundance of myofibroblasts was positively correlated with tumor growth and nodal metastasis in gastric carcinoma (P = 0.03, P = 0.0008, respectively). CONCLUSIONS: This clinical study revealed that overexpression of THBS-1 in stromal myofibroblasts is associated with tumor growth and nodal metastasis in gastric carcinoma. THBS-1 may activate latent transforming growth factor-ß1 to stimulate fibroblasts to differentiate into myofibroblasts, though further studies are needed to validate this hypothesis. These results suggest that THBS-1 and myofibroblasts may serve as novel targets for strategies aimed at protection against and treatment of gastric carcinoma.
Asunto(s)
Biomarcadores de Tumor/análisis , Ganglios Linfáticos/patología , Miofibroblastos/química , Neoplasias Gástricas/química , Neoplasias Gástricas/patología , Trombospondina 1/análisis , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Metástasis Linfática , Masculino , Persona de Mediana Edad , Miofibroblastos/patología , Estadificación de Neoplasias , ARN Mensajero/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Estómago/química , Trombospondina 1/genética , Regulación hacia ArribaRESUMEN
Thrombospondin-1 plays an important role in cancer development and progression. This study investigated if a correlation exists between single-nucleotide polymorphisms (SNPs) in the Thrombospondin-1 gene (THBS1) and gastric cancer. We conducted a case-control study on a randomly recruited population of 283 patients and 283 healthy individuals from the city of Fuzhou in Southeast China. Individuals were genotyped for four SNPs (rs1478604 A>G, rs2228261 C>T, rs2292305 T>C, and rs3743125 C>T) in THBS1 using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. THBS1 genotypic distributions between the case and control groups were tested for correlations with cancer development. Comparisons between the case and control groups showed no significant differences in the genotypic distributions of rs1478604 A>G, rs2228261 C>T, and rs3743125 C>T. However, we found a statistically significant association between homozygous CC of THBS1 rs2292305 T>C and development of highly differentiated carcinoma (HDC). The rs1478604 A>G variant was found to be associated with invasion and lymph node metastasis in gastric cancer. After logistic regression and stratification analysis, rs1478604 A>G was more strongly associated with lymph node metastasis in HDC gastric cancer. The power to detect an effect for rs1478604 A>G in HDC was 90%. These findings indicate that the THBS1 rs1478604 A>G variant is linked with differential risks for gastric cancer nodal metastasis. These results support further investigation of THBS1 as a potential therapeutic target in gastric cancer.
Asunto(s)
Carcinoma/genética , Carcinoma/secundario , Predisposición Genética a la Enfermedad/genética , Polimorfismo de Nucleótido Simple/genética , Neoplasias Gástricas/patología , Trombospondina 1/genética , Estudios de Casos y Controles , China , Genotipo , Humanos , Modelos Logísticos , Metástasis Linfática , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
PA28ß is a subunit of proteasome activator PA28. Previous study suggests that PA28ß is involved in the invasiveness and metastasis of gastric adenocarcinoma (GA), however, the mechanism is not fully understood. In the present study, we showed that invasive abilities of gastric cancer cells were enhanced when PA28ß being down-regulated, and were inhibited when PA28ß being overexpressed. To explore the possible mechanism of PA28ß associated elevated invasiveness, the protein profiles of PA28ß knock down and parental negative control gastric cancer cells were compared using proteomics approach. The results revealed that there were 43 proteins were differentially expressed, among them, chloride intracellular channel 1 (CLIC1) was significantly up-regulated and selected for further functional study. Down-regulation of CLIC1 by RNA interference was able to markedly inhibit cell invasion of PA28ß knock down gastric carcinoma cells. In addition, an inverse correlation between PA28ß and CLIC1 expressions was also verified in GA tissue samples, suggesting that knockdown of PA28ß could enhance tumor invasion and metastasis, at least in part, through up-regulation of CLIC1. Our results provide novel insight into the mechanisms of PA28ß related invasiveness and metastasis of GA, and suggest new alternative approaches for GA treatment.
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Canales de Cloruro/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Adulto , Anciano , Secuencia de Bases , Línea Celular Tumoral , Canales de Cloruro/genética , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/genética , Invasividad Neoplásica/fisiopatología , Complejo de la Endopetidasa Proteasomal/genética , Inhibidores de Proteasoma , Análisis por Matrices de Proteínas , Proteómica , Interferencia de ARN , ARN Interferente Pequeño/genética , Neoplasias Gástricas/genéticaRESUMEN
Snake venom cystatin (sv-cystatin) is a member of the cystatin family of cysteine protease inhibitors. To further evaluate the possibility of sv-cystatin in cancer therapy, this study examined the effects of sv-cystatin on the invasion and metastasis of liver cancer cells (MHCC97H) in vitro and in vivo as well as the underlying mechanism. sv-cystatin cDNA was transfected into MHCC97H cells and the anti-invasion and antimetastasis effects of sv-cystatin were determined using migration and matrigel invasion assays and a lung-metastasis mice model. The results suggest that sv-cyst clone (sv-cystatin expression in MHCC97H cells) delayed the invasion and metastasis in vitro and in vivo compared to the parental, mock and si-sv-cyst clone cells (inhibited sv-cystatin expression by siRNA). The decreased activities of cathepsin B, MMP-2 and MMP-9 and EMT change index including higher E-cadherin, lower N-cadherin and decreased Twist activity were observed in the sv-cyst clone, which contributes to the change in invasion and metastasis ability of MHCC97H cells. This study provides evidence that expression of the sv-cystatin gene in MHCC97H cells inhibits tumor cell invasion and metastasis through the reduction of the proteinases activity and Epithelial-Mesenchymal Transition (EMT), which might contribute to the anticancer research of the sv-cystatin protein.
Asunto(s)
Carcinoma Hepatocelular/terapia , Catepsina B/metabolismo , Cistatinas/metabolismo , Transición Epitelial-Mesenquimal , Gelatinasas/metabolismo , Neoplasias Hepáticas/terapia , Venenos de Serpiente/metabolismo , Animales , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/secundario , Línea Celular Tumoral , Movimiento Celular , Células Clonales , Cistatinas/antagonistas & inhibidores , Cistatinas/genética , Silenciador del Gen , Terapia Genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Masculino , Ratones , Ratones Desnudos , Invasividad Neoplásica/prevención & control , Proteínas Nucleares/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño , Proteína 1 Relacionada con Twist/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Studies have shown that expression of snake venom cystatin (sv-cystatin) in mouse melanoma cells and human gastric carcinoma cells can inhibit their invasion and metastasis. To advance the research into the biological features and pharmaceutical applications of sv-cystatin, we investigated the expression of recombinant sv-cystatin in an optimized Pichia pastoris system. Approximately 5 mg/L of bioactive sv-cystatin was obtained with a purity of 95.08%. Kinetic analyses of recombinant sv-cystatin revealed highly effective inhibitory efficiency against papain (Ki = 2.67 nM). We further investigated the effects of recombinant sv-cystatin on the invasion and metastasis of B16F10 cells and MHCC97H cells in vitro and in vivo. Matrigel invasion assays showed significant inhibition of recombinant sv-cystatin on the tumor cells in vitro. For experimental lung colonization assays, C57BL/6 mice inoculated in the lateral tail vein with B16F10 cells were treated with three i.v. injections of recombinant sv-cystatin (25 and 50 mg/kg) 24 h before cell inoculation, and 2 h and 24 h after cell inoculation. Administration of recombinant sv-cystatin significantly suppressed the formation of lung tumor colonies. For spontaneous metastasis assays, MHCC97H cells were inoculated s.c. into nude mice. After 24 h, recombinant sv-cystatin was administered by i.p. injections at 25, 50 or 100 mg/kg once daily for 5 days. Administration of recombinant sv-cystatin significantly decreased the formation of lung tumor colonies. Taken together, recombinant sv-cystatin inhibits the invasion and metastasis of tumor cells in vitro and in vivo. These results may facilitate the future evaluation of the pharmaceutical applications of sv-cystatin.
Asunto(s)
Cistatinas/metabolismo , Venenos Elapídicos/química , Invasividad Neoplásica/prevención & control , Metástasis de la Neoplasia/prevención & control , Proteínas Recombinantes/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colágeno , Cistatinas/análisis , Cistatinas/farmacología , Combinación de Medicamentos , Electroforesis en Gel de Poliacrilamida , Humanos , Immunoblotting , Cinética , Laminina , Ratones , Ratones Endogámicos C57BL , Papaína/antagonistas & inhibidores , Pichia , Proteoglicanos , Proteínas Recombinantes/farmacologíaRESUMEN
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RESUMEN
Hepatitis B spliced protein (HBSP) encoded by a 2.2 kb singly spliced hepatitis B virus (HBV) pre-genomic RNA (spliced between positions 2447 and 489 nt) is involved in the pathogenesis of HBV infection, whereas the exact mechanism is far from being fully elucidated. In this study, a yeast two-hybrid system using HBSP as bait was employed to screen binding partners for HBSP from a human liver cDNA library. The interaction between HBSP and fibrinogen γ chain (FGG) was further confirmed in vitro using a GST pull-down assay and confirmed in vivo using a mammalian two-hybrid assay and co-immunoprecipitation. It was identified that this interaction is mediated by the N terminal 47 amino acid residues of HBSP. HBSP could inhibit fibrin polymerization, factor XIIIa-mediated fibrin cross-linking, adhesion of platelets to fibrinogen and ADP-stimulated platelet aggregation. However, the interaction-mediating fragment 1-47 of HBSP is not sufficient for the inhibitory activity on fibrinogen function. The findings suggested that HBSP may participate in the hemostatic abnormality in patients with HBV-related liver diseases.
Asunto(s)
Fibrina/metabolismo , Fibrinógeno/metabolismo , Virus de la Hepatitis B/patogenicidad , Empalme del ARN , Proteínas Virales/metabolismo , Adulto , Secuencia de Aminoácidos , Línea Celular Tumoral , Factor XIIIa/metabolismo , Biblioteca de Genes , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , Humanos , Hígado/metabolismo , Datos de Secuencia Molecular , Adhesividad Plaquetaria , Agregación Plaquetaria/efectos de los fármacos , Técnicas del Sistema de Dos Híbridos , Proteínas Virales/química , Proteínas Virales/genética , Proteínas Virales/farmacologíaRESUMEN
A MYCL1 single nucleotide polymorphism, rs3134613, has been reported to play an important role in many cancers. However, its involvement in gastric cancer is controversial. The aim of the study was to investigate the influence of rs3134613 on the development and progression of gastric cancer in a southeast Chinese population. Genotypes were analyzed by polymerase chain reaction-restriction fragment length polymorphism in 317 gastric cancer patients and 200 cancer-free controls. Data show that the risk of diffuse-type gastric cancer in carriers with the T allele (T/T or G/T genotype) was higher than that in carriers with the G/G genotype (adjusted odds ratio [OR] = 2.601, 95% confidence interval [CI] = 1.431-4.895, p = 0.003). The risk of diffuse-type gastric cancer in T allele carriers was higher than that in G allele carriers (adjusted OR = 1.594, 95% CI = 1.157-2.286, p = 0.009). The risk of poorly differentiated cancer in carriers with the T allele (T/T or G/T genotype) was higher than that in carriers with the G/G genotype (adjusted OR = 1.963, 95% CI = 1.156-3.325, p = 0.015). The results demonstrate that rs3134613 is associated with susceptibility to diffuse-type gastric cancer and with differentiation of gastric cancer. rs3134613 may be used as a potential marker to identify individuals who are at high risk of diffuse-type gastric cancer.
Asunto(s)
Predisposición Genética a la Enfermedad , Proteínas Proto-Oncogénicas c-myc/genética , Neoplasias Gástricas/genética , Alelos , Pueblo Asiatico/genética , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Polimorfismo de Nucleótido SimpleRESUMEN
The 2.2 kb doubly spliced defective hepatitis B virus (HBV) genome is frequently detected in the serum of patients with chronic hepatitis B. However, the biological significance of this type of defective genome is not well understood. In this study, expression of the hepatitis B doubly spliced protein (HBDSP) was confirmed from the 2.2 kb doubly spliced defective HBV genome, which was isolated and transfected into Huh-7 hepatoma cells. To explore the potential pathogenicity of HBDSP, hepatocellular proteins interacting with HBDSP were screened by a yeast two-hybrid assay. Unexpectedly, HBDSP could transactivate the GAL4-responsive element, and deletion mapping revealed that the fragment located between residues Leu-48 and Gln-75 of HBDSP was crucial for transactivation activity. In Huh-7 hepatoma cells, HBDSP localized predominantly to the cytoplasm and showed transactivating effects on the cytomegalovirus immediate-early promoter, simian virus 40 enhancer/promoter and HBV regulatory elements including the S1 promoter, S2 promoter, Enhancer I and core upstream regulatory sequences. Further studies revealed that the transactivating activities were mediated by activator protein-1- and CCAAT/enhancer-binding protein-binding sites. These findings suggest that HBDSP is a pleiotropic activator protein that can potentially serve as an HBV virulence factor.
Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Regulación Viral de la Expresión Génica , Virus de la Hepatitis B/patogenicidad , Empalme del ARN , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Factor de Transcripción AP-1/metabolismo , Animales , Sitios de Unión , Línea Celular , Hepatitis B Crónica/virología , Hepatocitos/virología , Humanos , Datos de Secuencia Molecular , Unión Proteica , Transactivadores/metabolismo , Técnicas del Sistema de Dos HíbridosRESUMEN
Advanced glycation end products (AGEs) and their interaction with the receptor for advanced glycation end products (RAGE) play an important role in diabetic vascular complications. The current study demonstrated that AGEs significantly increased RAGE expression and the release of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) in human umbilical vein endothelial cell-derived line ECV304 cells. RAGE antisense RNA partially inhibited the expression of TNF-alpha and IL-6 induced by AGEs. Oligonucleotide microarray was used to identify the genes that respond to RAGE activation. Phospholipase C beta 1 (PLC beta 1), phospholipase C beta 4 (PLC beta 4) and calcium/calmodulin-dependent protein kinase IV (CAMK IV) which associated with Ca(2+) signaling were upregulated. The rise of intracellular calcium and the NF-kappaB promoter activity induced by AGEs were suppressed by RAGE antisense RNA, PLC inhibitor U73122 and dominant negative CAMK IV, respectively. These findings suggest that PLC/CAMK IV-NF-kappaB is involved in RAGE mediated signaling pathway in human endothelial cells.
Asunto(s)
Proteína Quinasa Tipo 4 Dependiente de Calcio Calmodulina/metabolismo , Células Endoteliales/enzimología , FN-kappa B/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/metabolismo , Calcio/metabolismo , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Endoteliales/citología , Células Endoteliales/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Genes Dominantes , Productos Finales de Glicación Avanzada/farmacología , Humanos , Interleucina-6/biosíntesis , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , ARN sin Sentido/metabolismo , Receptor para Productos Finales de Glicación Avanzada , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Fosfolipasas de Tipo C/antagonistas & inhibidoresRESUMEN
PURPOSE: This study aimed to investigate the expression level of human proteasome activator PA28beta subunit (PA28beta) in gastric adenocarcinomas (GA) tissues and investigate its potential role in GA carcinogenesis. METHODS: To investigate the expression profile of PA28beta in patients with GA, we employed immunohistochemistry for detection of 287 cases of paired GA and adjacent non-neoplastic tissues. To evaluate the role of PA28beta in GA cells, we measured cell growth, colony formation, soft agar, and nude mice tumorigenicity assays in MKN-45 GA cells pre- and post-PA28beta transfection. RESULTS: PA28beta had lower expression in 183 of 287 GA cases compared to paired normal samples (63.76%; P < or = 0.001). Decreased expression was dependent on histological type, TNM stage, and differentiation grade. Significantly decreased expression was correlated with a diffuse histological type (88/116, 75.86%) compared to an intestinal type (84/152, 55.26%; P < or = 0.001), with advanced TNM stages (T3: 44/59, 74.58%; T4:25/32, 78.13%) compared to earlier stages (T1: 25/47, 53.19%; T2: 90/149, 60.40%; P = 0.004), and poorer differentiation grade (poor: 68/90, 75.56%) compared to a higher grade (high: 9/18, 50%, moderate: 74/134, 55.22%) (P = 0.006). Over-expression of PA28beta inhibited cell growth, proliferation, and tumorigenicity of MKN-45 GA cells. CONCLUSIONS: These results indicated that PA28beta might participate in the origin and progression of GA cancer through changes to cell proliferation activity and tumorigenicity. Therefore, PA28beta might be a novel biomarker for GA.