RESUMEN
BACKGROUND: The risk of hepatocellular carcinoma (HCC) increases in chronic hepatitis B surface antigen (HBsAg) carriers who often have concomitant increase in the levels of benzo[alpha]pyrene-7,8-diol-9,10-epoxide(±) (BPDE)-DNA adduct in liver tissues, suggesting a possible co-carcinogenesis of Hepatitis B virus (HBV) and benzo[alpha]pyrene in HCC; however the exact mechanisms involved are unclear. METHODS: The interaction between hepatitis B spliced protein (HBSP) and microsomal epoxide hydrolase (mEH) was confirmed using GST pull-down, co-immunoprecipitation and mammalian two-hybrid assay; the effects of HBSP on mEH-mediated B[alpha]P metabolism was examined by high performance liquid chromatography (HPLC); and the influences of HBSP on B[alpha]P carcinogenicity were evaluated by bromodeoxyuridine cell proliferation, anchorage-independent growth and tumor xenograft. RESULTS: HBSP could interact with mEH in vitro and in vivo, and this interaction was mediated by the N terminal 47 amino acid residues of HBSP. HBSP could greatly enhance the hydrolysis activity of mEH in cell-free mouse liver microsomes, thus accelerating the metabolism of benzo[alpha]pyrene to produce more ultimate carcinnogen, BPDE, and this effect of HBSP requires the intact HBSP molecule. Expression of HBSP significantly increased the formation of BPDE-DNA adduct in benzo[alpha]pyrene-treated Huh-7 hepatoma cells, and this enhancement was blocked by knockdown of mEH. HBSP could enhance the cell proliferation, accelerate the G1/S transition, and promote cell transformation and tumorigenesis of B[alpha]P-treated Huh-7 hepatoma cells. CONCLUSIONS: Our results demonstrated that HBSP could promote carcinogenic effects of B[alpha]P by interacting with mEH and enhancing its hydrolysis activity.
Asunto(s)
Carcinogénesis , Carcinoma Hepatocelular/genética , Epóxido Hidrolasas/metabolismo , Neoplasias Hepáticas/genética , Proteínas Virales/metabolismo , Animales , Benzopirenos/toxicidad , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Aductos de ADN/metabolismo , Epóxido Hidrolasas/genética , Regulación Viral de la Expresión Génica , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/patogenicidad , Humanos , Hidrólisis/efectos de los fármacos , Neoplasias Hepáticas/patología , Ratones , Microsomas/efectos de los fármacos , Microsomas/enzimología , Proteínas Virales/genéticaRESUMEN
Hepatitis B spliced protein (HBSP) is involved in the pathogenicity and/or persistence of hepatitis B virus (HBV). Chronic HBV infection is one of the most important risk factors for the development of hepatocellular carcinoma (HCC). However, whether or not HBSP contributes to the progression of HBV-associated HCC remains unknown. This study reports that overexpression of HBSP in human hepatoma cells increased cell invasion and motility. Conversely, small interfering RNA (siRNA)-mediated knockdown of HBSP expression inhibited migration and invasion. By glutathione S-transferase (GST) pulldown, coimmunoprecipitation, and a mammalian two-hybrid assay, HBSP was found to directly interact with cathepsin B (CTSB). Similar to HBSP knockdown, knocking down CTSB also reduced cell migration and invasion. Furthermore, the HBSP-overexpressing hepatoma cells were shown to have increased expression and activity of matrix metalloproteinase-9 (MMP-9) and urokinase-type plasminogen activator (uPA), and overexpression of HBSP significantly enhanced tumor-induced vascularization of endothelial cells. In contrast, knockdown of either HBSP or CTSB by siRNA resulted in inhibition of the two proteolytic enzymes and of the in vitro angiogenesis. Expression of HBSP in the hepatoma cells appeared to activate the mitogen-activated protein kinase (MAPK) and Akt signaling pathway, as evidenced by increases in phosphorylation of p38, Jun N-terminal protein kinase (JNK), extracellular signal-regulated kinase (ERK), and Akt. Taken together, these findings imply that interaction of HBSP with CTSB may promote hepatoma cell motility and invasion and highlight new molecular mechanisms for HBSP-induced HCC progression that involve the secretion and activation of proteolytic enzymes, increased tumor-induced angiogenesis, and activation of the MAPK/Akt signaling, thereby leading to the aggressiveness of hepatoma cells.
Asunto(s)
Carcinoma Hepatocelular/patología , Catepsina B/metabolismo , Neoplasias Hepáticas/patología , Invasividad Neoplásica , Metástasis de la Neoplasia , Proteínas Virales/metabolismo , Secuencia de Bases , Carcinoma Hepatocelular/irrigación sanguínea , Línea Celular Tumoral , Cartilla de ADN , Humanos , Inmunoprecipitación , Neoplasias Hepáticas/irrigación sanguínea , Neovascularización Patológica , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Hepatitis B virus (HBV)-encoded X protein (HBx protein) is a multi-functional regulatory protein. It functions by protein-protein interaction and plays a pivotal role in the pathogenesis of HBV-related diseases. However, the partners in hepatocytes interacting with HBx protein are far from understood fully. In this study, immunoprecipitation was employed to screen for binding partners for the HBx protein from huh-7 hepatoma cells infected with recombinant adenovirus expressing HBx protein, and five cellular proteins including eukaryotic translation elongation factor 1 alpha 1 (eEF1A1), were identified. The interaction between HBx protein and eEF1A1 was confirmed further using a GST pull-down assay and co-immunoprecipitation, respectively. In Huh-7 hepatoma cells, the HBx protein inhibits dimer formation of eEF1A1, hence blocks filamentous actin bundling. These findings provide new insights into the molecular mechanisms involved in the functions of the HBx protein.
Asunto(s)
Actinas/antagonistas & inhibidores , Virus de la Hepatitis B/patogenicidad , Interacciones Huésped-Patógeno , Factor 1 de Elongación Peptídica/metabolismo , Multimerización de Proteína , Transactivadores/metabolismo , Adenoviridae/genética , Línea Celular , Vectores Genéticos , Hepatocitos/virología , Humanos , Inmunoprecipitación , Unión Proteica , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Hepatitis B virus X protein (HBx protein) is a multifunctional regulatory protein. The transactivation of nuclear factor kappa B (NF-κB) by HBx protein has been shown to be of importance in the pathogenesis of HBV-related diseases. However, the mechanism involved remains largely unclear. In this study, a CytoTrap yeast two-hybrid system was employed to screen binding partners of the HBx protein; 29 cellular proteins, including valosin-containing protein (VCP), were identified. The interaction between HBx protein and VCP was further confirmed in vitro and in vivo using a glutathione S-transferase pull-down assay and co-immunoprecipitation, respectively. It was also shown that this interaction is mediated by amino acid residues 51-120 of the HBx protein. In Huh-7 hepatoma cells, HBx protein enhanced the VCP-mediated activation of NF-κB. Our findings provide new insights into the molecular mechanisms that lead to the activation of NF-κB by HBx protein.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Virus de la Hepatitis B/metabolismo , Hepatitis B/metabolismo , FN-kappa B/genética , Transactivadores/metabolismo , Activación Transcripcional , Adenosina Trifosfatasas/genética , Secuencias de Aminoácidos , Proteínas de Ciclo Celular/genética , Línea Celular , Hepatitis B/virología , Virus de la Hepatitis B/química , Virus de la Hepatitis B/genética , Humanos , FN-kappa B/metabolismo , Unión Proteica , Transactivadores/química , Transactivadores/genética , Proteína que Contiene Valosina , Proteínas Reguladoras y Accesorias ViralesRESUMEN
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
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
Based on analysis of Epstein-Barr virus (EBV) BART microRNA expression profiles, we previously reported that EBV-encoded miR-BART13 is upregulated in nasopharyngeal carcinoma (NPC) plasma specimens. However, the effects and molecular mechanisms of miR-BART13 in NPC remain largely unknown. We found that miR-BART13 was significantly upregulated in NPC tissue specimens. Ectopic expression of miR-BART13 promoted NPC cell proliferation, epithelial mesenchymal transition, and metastasis in vitro, and facilitated xenograft tumor growth and lung metastasis in vivo. Molecularly, NF-κB inhibitor interacting Ras-like 2 (NKIRAS2), a negative regulator of the NF-κB signaling, was identified to be a direct target of miR-BART13 in NPC cells, and NKIRAS2 mRNA and protein expression was inversely correlated with miR-BART13 in NPC tissues, respecitvely. Furthermore, the NF-κB signaling pathway was activated by miR-BART13. By rescued experiments, reconstitution of NKIRAS2 expression abrogated all the phenotypes upregulated by miR-BART13, and attenuated activity of NF-κB signaling pathway activated by miR-BART13 in NPC cells. Our findings indicated the newly identified miR-BART13/NKIRAS2/NF-κB signaling axis may provide further insights into better understanding of NPC initiation and development, and targeting of this pathway could be further studied as a therapeutic strategy for NPC patients.
Asunto(s)
Proliferación Celular/genética , Herpesvirus Humano 4/genética , MicroARNs/genética , Carcinoma Nasofaríngeo/virología , Metástasis de la Neoplasia/genética , ARN Viral/genética , Transducción de Señal/genética , Animales , Proteínas Portadoras/genética , Línea Celular Tumoral , Infecciones por Virus de Epstein-Barr/genética , Infecciones por Virus de Epstein-Barr/virología , Femenino , Regulación Neoplásica de la Expresión Génica/genética , Células HEK293 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , FN-kappa B/genética , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Neoplasias Nasofaríngeas/virología , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
In this study, we determine the relationship between the expression of major histocompatibility complex class I chain-related gene A (MICA) in gastric cancer tumors after D2 gastrectomy and the clinical outcome of a CIK-containing adjuvant therapy. Ninety-five consecutive patients with gastric cancer after D2 gastrectomy who received adjuvant chemotherapy combined with CIK cell therapy were enrolled. The MICA expression of their tumors was determined by immunohistochemistry (IHC). High expression of MICA protein was documented by IHC in 38 of 95 tumor samples (40.0 %). The MICA status was significantly associated with the age and stage, p = 0.008 and 0.023, respectively. Analysis of NKG2D on in vitro expanded CIK cells showed that the percentages of NKG2D+ in CD3+/CD56+, CD3-/CD56+, and CD3+/CD8+ cells populations were 97.2 ± 1.4, 97.9 ± 1.8, and 95.6 ± 2.1 %, respectively. For patient with high MICA-expressing tumors, the median DFS and OS were longer than for the patients with tumors with low expression of MICA; 46.0 versus 41.0 months (p = 0.027), and 48.0 versus 42.0 months (p = 0.031), respectively. In a multivariate analysis, stage and MICA expression were independent prognostic factors for DFS and OS. Our findings show that adjuvant chemotherapy plus CIK therapy treatment is a promising modality for treating gastric cancer patients after D2 gastrectomy. Especially, those who have tumors with high expression of MICA were more likely to benefit from such a treatment strategy. Subsequent studies in clinical trial cohorts will be required to confirm the clinical utility of these markers.
Asunto(s)
Carcinoma/terapia , Células Asesinas Inducidas por Citocinas/trasplante , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunoterapia/métodos , Neoplasias Gástricas/terapia , Factores de Edad , Anciano , Biomarcadores/metabolismo , Carcinoma/mortalidad , Carcinoma/patología , Quimioterapia Adyuvante , Terapia Combinada , Células Asesinas Inducidas por Citocinas/inmunología , Femenino , Gastrectomía , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Masculino , Estadificación de Neoplasias , Pronóstico , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
Hepatitis B virus X protein (HBx) transactivates multiple transcription factors including nuclear factor-kappa B (NF-κB) that regulates inflammatory-related genes. However, the regulatory mechanism of HBx in NF-κB activation remains largely unknown. This study reports that HBx augments the interleukin-1ß (IL-1ß)-induced NF-κB activation via interaction with a Toll-like receptor (TLR) adapter protein, ECSIT (evolutionarily conserved signaling intermediate in Toll pathways). GST pull-down and co-immunoprecipitation analyses showed that HBx interacted with ECSIT. Deletion analysis of HBx in a CytoTrap two-hybrid system revealed that the interaction region of HBx for ECSIT was attributed to aa 51-80. Co-transfection of HBx and ECSIT in IL-1ß-stimulated cells appeared to activate IKK and IκB signaling pathway as phosphorylation of both IKK α/ß and IκBα was increased whereas knockdown of ECSIT or HBxΔ51-80 mutant attenuated the phosphorylation. As a consequence of IκBα degradation, NF-κB was activated as evidenced by increases in NF-κB transcriptional activity and the nuclear translocation of p65 and p50 that resulted in the induction of IL-10. In contrast, knockdown of ECSIT by siRNA or treatment with an NF-κB selective inhibitor (helenalin) abolished the NF-κB activation and IL-10 expression. We conclude that ECSIT appears to be a novel HBx-interacting signal molecule and their interaction is mechanistically important in IL-1ß induction of NF-κB activation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Virus de la Hepatitis B/inmunología , Interacciones Huésped-Patógeno , Interleucina-1beta/metabolismo , FN-kappa B/metabolismo , Transducción de Señal , Transactivadores/metabolismo , Línea Celular , Análisis Mutacional de ADN , Humanos , Unión Proteica , Mapeo de Interacción de Proteínas , Técnicas del Sistema de Dos Híbridos , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Cytokine-induced killer (CIK) cells have shown promising activity against gastric cancer in vitro and in vivo. Previous studies showed that cell signaling through MHC I-related Chain A (MICA)-Natural killer group 2, member D (NKG2D) results in CIK cell activation leading to cytolytic activities against tumor cells. In this study, we investigate the MICA status in patients with gastric carcinoma, and determine the potential relationship between MICA and clinical outcome of a CIK containing therapy. Two hundred and forty-three patients with gastric cancer who had received curative D2 gastrectomy were enrolled. The MICA expression of their tumors was determined by immunohistochemistry (IHC). Disease-free survival (DFS) and overall survival (OS) were evaluated. One hundred and forty-eight patients received adjuvant chemotherapy alone, and 95 patients received adjuvant chemotherapy combined with autologous CIK cell therapy. Patients who received adjuvant chemotherapy plus CIK had significantly longer DFS, 42.0 months vs. 32.0 months (P = 0.012), and OS, 45.0 months vs. 42.0 months (P = 0.039), by log-rank test. MICA high-expression, IHC scores of 5-7, was found in tumors from 89 of 243 patients (36.6%). The MICA expression was significantly correlated with the stage (P = 0.007) and there was a borderline association with histological grade (P = 0.054). In the adjuvant chemotherapy plus CIK group (n = 95), patients with high MICA expression had longer DFS, 46.0 months vs. 41.0 months (P = 0.027), and OS, 48.0 months vs. 42.0 months (P = 0.031). In the adjuvant chemotherapy alone group (n = 148), the median DFS and OS had no significant correlation with the MICA status. In a multivariate analysis stage, CIK therapy, and the interaction of MICA status and CIK therapy were independent prognostic factors for DFS and OS. Our study indicated that adjuvant chemotherapy plus CIK immunotherapy is a promising modality for treating gastric cancer patients after D2 gastrectomy. MICA status was associated with the outcome measures in CIK therapy, validation in prospective clinical trials is required to assess the value of this biomarker in the clinical decision-making process.