RESUMEN
DNA-reactive compounds are harnessed for cancer chemotherapy. Their genotoxic effects are considered to be the main mechanism for the cytotoxicity to date. Because this mechanism preferentially affects actively proliferating cells, it is postulated that the cytotoxicity is specific to cancer cells. Nonetheless, they do harm normal quiescent cells, suggesting that there are other cytotoxic mechanisms to be uncovered. By employing doxorubicin as a representative DNA-reactive compound, we have discovered a cytotoxic mechanism that involves a cellular noncoding RNA (ncRNA) nc886 and protein kinase R (PKR) that is a proapoptotic protein. nc886 is transcribed by RNA polymerase III (Pol III), binds to PKR, and prevents it from aberrant activation in most normal cells. We have shown here that doxorubicin evicts Pol III from DNA and, thereby, shuts down nc886 transcription. Consequently, the instantaneous depletion of nc886 provokes PKR and leads to apoptosis. In a short-pulse treatment of doxorubicin, these events are the main cause of cytotoxicity preceding the DNA damage response in a 3D culture system as well as the monolayer cultures. By identifying nc886 as a molecular signal for PKR to sense doxorubicin, we have provided an explanation for the conundrum why DNA-damaging drugs can be cytotoxic to quiescent cells that have the competent nc886/PKR pathway.
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Apoptosis/efectos de los fármacos , ADN/metabolismo , MicroARNs/metabolismo , ARN no Traducido , Línea Celular , Doxorrubicina/farmacología , Humanos , MicroARNs/genética , ARN Polimerasa III/metabolismo , ARN no Traducido/genética , ARN no Traducido/metabolismo , Transducción de Señal/efectos de los fármacos , eIF-2 Quinasa/metabolismoRESUMEN
Interferons (IFNs) are a crucial component in the innate immune response. Especially the IFN-ß signaling operates in most cell types and plays a key role in the first line of defense upon pathogen intrusion. The induction of IFN-ß should be tightly controlled, because its hyperactivation can lead to tissue damage or autoimmune diseases. Activation of the IFN-ß promoter needs Interferon Regulatory Factor 3 (IRF3), together with Nuclear Factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Activator Protein 1 (AP-1). Here we report that a human noncoding RNA, nc886, is a novel suppressor for the IFN-ß signaling and inflammation. Upon treatment with several pathogen-associated molecular patterns and viruses, nc886 suppresses the activation of IRF3 and also inhibits NF-κB and AP-1 via inhibiting Protein Kinase R (PKR). These events lead to decreased expression of IFN-ß and resultantly IFN-stimulated genes. nc886's role might be to restrict the IFN-ß signaling from hyperactivation. Since nc886 expression is regulated by epigenetic and environmental factors, nc886 might explain why innate immune responses to pathogens are variable depending on biological settings.
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Regulación de la Expresión Génica/inmunología , Factor 3 Regulador del Interferón/inmunología , Interferón Tipo I/inmunología , ARN no Traducido/inmunología , Animales , Línea Celular Tumoral , Células HCT116 , Células HEK293 , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad Innata/genética , Inmunidad Innata/inmunología , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Interferón Tipo I/genética , Interferón Tipo I/metabolismo , Ratones , FN-kappa B/inmunología , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Células RAW 264.7 , ARN no Traducido/genética , Transducción de Señal/inmunología , Factor de Transcripción AP-1/inmunología , Factor de Transcripción AP-1/metabolismo , Virus/inmunología , eIF-2 Quinasa/genética , eIF-2 Quinasa/inmunología , eIF-2 Quinasa/metabolismoRESUMEN
Antibody drug conjugates (ADCs), consisting of a cancer-specific antibody and cytotoxic payload, are shown to be a potent class of anticancer therapeutics, with enhanced therapeutic efficacy and reduced "off-target" side effects. However, the therapeutic window of ADCs is narrowed by problems such as difficulty in site-specific conjugation of payload, changes in antibody stability due to payload conjugation, and difficulty in tissue penetration. In this respect, aptamers have advantages in drug-delivery, as they can be easily and stably conjugated with cytotoxic drugs. We previously reported that oligobody, an aptamer-antibody complex, is a novel delivery method for aptamer-based therapeutics. In the current study, we describe DOligobody, a drug-conjugated oligobody comprising an aptamer-drug conjugate and an antibody. A cotinine-conjugated anti-HER2 aptamer (cot-HER2apt) was specifically bound to HER2-positive NCI-N87 cells, and underwent receptor-mediated endocytosis. Further, HER2-DOligobody, a cot-HER2apt-conjugated monomethyl auristatin E (cot-HER2apt-MMAE) oligobody, inhibited the growth of HER2-positive NCI-N87 cells. Finally, systemic administration of HER2-DOligobody significantly reduced tumor growth in a xenograft mouse model. Taken together, these results suggest that our DOligobody strategy may be a powerful platform for rapid, low-cost and effective cancer therapy.
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Inmunoconjugados/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Receptor ErbB-2/inmunología , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Aptámeros de Péptidos/química , Línea Celular Tumoral , Proliferación Celular , Cotinina/química , Endocitosis , Femenino , Humanos , Inmunoconjugados/química , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Oligopéptidos/químicaRESUMEN
TopBP1 was initially identified as a topoisomerase II-ß-binding protein and it plays roles in DNA replication and repair. We found that TopBP1 is expressed at high levels in lymphoid tissues and is essential for early lymphocyte development. Specific abrogation of TopBP1 expression resulted in transitional blocks during early lymphocyte development. These defects were, in major part, due to aberrant V(D)J rearrangements in pro-B cells, double-negative and double-positive thymocytes. We also show that TopBP1 was located at sites of V(D)J rearrangement. In TopBP1-deficient cells, γ-H2AX foci were found to be increased. In addition, greater amount of γ-H2AX product was precipitated from the regions where TopBP1 was localized than from controls, indicating that TopBP1 deficiency results in inefficient DNA double-strand break repair. The developmental defects were rescued by introducing functional TCR αß transgenes. Our data demonstrate a novel role for TopBP1 as a crucial factor in V(D)J rearrangement during the development of B, T and iNKT cells.
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Proteínas Portadoras/genética , Reparación del ADN , ADN/genética , Linfocitos/fisiología , Recombinación V(D)J/inmunología , Animales , Proteínas Portadoras/inmunología , Proteínas Portadoras/metabolismo , Inmunoprecipitación de Cromatina , Roturas del ADN de Doble Cadena , Daño del ADN , Expresión Génica , Histonas/genética , Histonas/metabolismo , Linfocitos/inmunología , Ratones , Ratones Noqueados , Células T Asesinas Naturales/inmunología , Células T Asesinas Naturales/fisiología , Células Precursoras de Linfocitos B/inmunología , Células Precursoras de Linfocitos B/fisiología , Eliminación de Secuencia , Organismos Libres de Patógenos Específicos , Transgenes , Recombinación V(D)J/genéticaRESUMEN
Malignant brain tumor mass contains significant numbers of infiltrating glial cells that may intimately interact with tumor cells and influence cancer treatments. Understanding of characteristic discrepancies between normal GLIA and tumor cells would, therefore, be valuable for improving anticancer therapeutics. Here, we report distinct differences in toll-like receptors (TLR)-2-mediated responses between normal glia and primary brain tumor cell lines. We found that tyrosine phosphorylation of STAT1 by TLR2 ligands and its downstream events did not occur in mouse, rat, or human brain tumor cell lines, but were markedly induced in normal primary microglia and astrocytes. Using TLR2-deficient, interferon (IFN)-γ-deficient, and IFNγ-receptor-1-deficient mice, we revealed that the impaired phosphorylation of STAT1 might be linked with defective TLR2 system in tumor cells, and that a TLR2-dependent pathway, not IFNγ-receptor machinery, might be critical for tyrosine STAT1 phosphorylation by TLR2 ligands. We also found that TLR2 and its heterodimeric partners, TLR1 and 6, on brain tumor cells failed to properly respond to TLR2 ligands, and representative TLR2-dependent cellular events, such as inflammatory responses and cell death, were not detected in brain tumor cells. Similar results were obtained in in vitro and in vivo experiments using orthotopic mouse and rat brain tumor models. Collectively, these results suggest that primary brain tumor cells may exhibit a distinctive dysfunction of TLR2-associated responses, resulting in abnormal signaling and cellular events. Careful targeting of this distinctive property could serve as the basis for effective therapeutic approaches against primary brain tumors.
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Neoplasias Encefálicas/patología , Neuroblastoma/patología , Neuroglía/metabolismo , Receptor Toll-Like 2/metabolismo , Animales , Animales Recién Nacidos , Línea Celular Tumoral , Células Cultivadas , Corteza Cerebral/citología , Modelos Animales de Enfermedad , Interferón gamma , Ligandos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Fosforilación , Ratas , Ratas Sprague-Dawley , Receptores de Interferón/deficiencia , Receptores de Interferón/genética , Receptor Toll-Like 2/genética , Receptor de Interferón gammaRESUMEN
Blood metabolites can be detected as low-mass ions (LMIs) by mass spectrometry (MS). These LMIs may reflect the pathological changes in metabolism that occur as part of a disease state, such as cancer. We constructed a LMI discriminant equation (LOME) to investigate whether systematic LMI profiling might be applied to cancer screening. LMI information including m/z and mass peak intensity was obtained by five independent MALDI-MS analyses, using 1,127 sera collected from healthy individuals and cancer patients with colorectal cancer (CRC), breast cancer (BRC), gastric cancer (GC) and other types of cancer. Using a two-stage principal component analysis to determine weighting factors for individual LMIs and a two-stage LMI selection procedure, we selected a total of 104 and 23 major LMIs by the LOME algorithms for separating CRC from control and rest of cancer samples, respectively. CRC LOME demonstrated excellent discriminating power in a validation set (sensitivity/specificity: 93.21%/96.47%). Furthermore, in a fecal occult blood test (FOBT) of available validation samples, the discriminating power of CRC LOME was much stronger (sensitivity/specificity: 94.79%/97.96%) than that of the FOBT (sensitivity/specificity: 50.00%/100.0%), which is the standard CRC screening tool. The robust discriminating power of the LOME scheme was reconfirmed in screens for BRC (sensitivity/specificity: 92.45%/96.57%) and GC (sensitivity/specificity: 93.18%/98.85%). Our study demonstrates that LOMEs might be powerful noninvasive diagnostic tools with high sensitivity/specificity in cancer screening. The use of LOMEs could potentially enable screening for multiple diseases (including different types of cancer) from a single sampling of LMI information.
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Biomarcadores de Tumor/sangre , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/diagnóstico , Adulto , Anciano , Anciano de 80 o más Años , Algoritmos , Neoplasias de la Mama/sangre , Neoplasias de la Mama/diagnóstico , Detección Precoz del Cáncer/métodos , Femenino , Humanos , Masculino , Tamizaje Masivo/métodos , Persona de Mediana Edad , Sangre Oculta , Sensibilidad y Especificidad , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Neoplasias Gástricas/sangre , Neoplasias Gástricas/diagnóstico , Adulto JovenRESUMEN
PURPOSE: c-MYC is a promising target for cancer therapy but its use is restricted by unwanted, devastating side effects. We explored whether intravesical instillation of the c-MYC inhibitor KSI-3716 could suppress tumor growth in murine orthotopic bladder xenografts. MATERIALS AND METHODS: The small molecule KSI-3716, which blocks c-MYC/MAX binding to target gene promoters, was used as an intravesical chemotherapy agent. KSI-3716 action was assessed by electrophoretic mobility shift assay, chromatin immunoprecipitation, transcription reporter assay and quantitative reverse transcriptase-polymerase chain reaction. Inhibition of cell proliferation and its mechanism was monitored by cell cytotoxicity assay, EdU incorporation assay and flow cytometry. The in vivo efficacy of KSI-3716 was examined by noninvasive luminescence imaging and histological analysis after intravesical instillation of KSI-3716 in murine orthotopic bladder xenografts. RESULTS: KSI-3716 blocked c-MYC/MAX from forming a complex with target gene promoters. c-MYC mediated transcriptional activity was inhibited by KSI-3716 at concentrations as low as 1 µM. The expression of c-MYC target genes, such as cyclin D2, CDK4 and hTERT, was markedly decreased. KSI-3716 exerted cytotoxic effects on bladder cancer cells by inducing cell cycle arrest and apoptosis. Intravesical instillation of KSI-3716 at a dose of 5 mg/kg significantly suppressed tumor growth with minimal systemic toxicity. CONCLUSIONS: The c-MYC inhibitor KSI-3716 could be developed as an effective intravesical chemotherapy agent for bladder cancer.
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4-Quinolonas/antagonistas & inhibidores , Compuestos de Anilina/antagonistas & inhibidores , Antineoplásicos/administración & dosificación , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , 4-Quinolonas/administración & dosificación , Administración Intravesical , Compuestos de Anilina/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Western Blotting , Puntos de Control del Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Ensayo de Cambio de Movilidad Electroforética , Femenino , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética/efectos de los fármacos , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cytotoxic T lymphocyte-associated antigen 4 (CTLA4) has been known to be a strong tolerance-inducing inhibitory receptor on T-cell surface. Systemic blocking of CTLA4 function with blocking antibodies has been regarded as an attractive strategy to enhance antitumor immunity. However, this strategy accompanies systemic autoimmune side effects that are sometimes problematic. Therefore, we developed a novel CTLA4 mutant that could be expressed in tumor antigen-specific T cells to enhance antitumor effect without systemic autoimmunity. This mutant, named CTLA4-CD28 chimera, consists of extracellular and transmembrane domains of CTLA4, linked with cytoplasmic CD28 domain. Overexpression of CTLA4-CD28 chimera in T cells delivered stimulatory signals rather than inhibitory signals of CTLA4 and significantly enhanced T-cell reactivity. Although this effect was observed in both CD4 and CD8 T cells, the effect on CD4 T cells was predominant. CTLA4-CD28 chimera gene modification of CD4 T cells significantly enhanced antitumor effect of unmodified CD8 T cells. Nonetheless, the gene modification of CD8 T cells along with CD4 T cells further maximized antitumor effect of T cells in 2 different murine tumor models. Thus, CTLA4-CD28 chimera gene modification of both tumor antigen-specific CD4 and CD8 T cells would be an ideal way of modulating CTLA4 function to enhance tumor-specific T-cell reactivity.
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Traslado Adoptivo , Antígeno CTLA-4/inmunología , Neoplasias/inmunología , Neoplasias/terapia , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Antígenos de Neoplasias/inmunología , Antígenos CD28/genética , Antígenos CD28/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígeno CTLA-4/genética , Proliferación Celular , Modelos Animales de Enfermedad , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Células Jurkat , Linfoma/inmunología , Linfoma/terapia , Melanoma/inmunología , Melanoma/terapia , Ratones , Resultado del TratamientoRESUMEN
Adenoviruses harboring the herpes simplex virus thymidine kinase (HSVtk) gene under the regulation of a trans-splicing ribozyme targeting human telomerase reverse transcriptase (hTERT-TR) show marked and specific antitumor activity. In addition to inducing tumor cell death by direct cytotoxicity, it is becoming clear that HSVtk also induces antitumor immunity. Programmed death ligand 1 (PD-L1) expressed on tumor cell surfaces mediates tumor-induced immunoresistance by inhibiting PD1-expressing tumor-infiltrating T cells. Here, we explored whether a soluble form of PD1 (sPD1-Ig), which blocks PD-L1, could synergize with TERT-TR-regulated HSVtk to enhance the adenoviral therapeutic efficacy by boosting antitumor immunity. Tumor antigen released by HSVtk-transduced tumors successfully primed tumor antigen-specific CD8 T cells via dendritic cells (DC). Regression of murine tumors was markedly enhanced when sPD1-Ig was incorporated into the adenovirus as compared with a single-module adenovirus expressing only HSVtk. This effect was abolished by CD8 T-cell depletion. Consistent with this, following adoptive transfer of tumor antigen-specific CD8 T cells into tumor-bearing Rag1(-/-) mice, dual-module adenovirus significantly enhanced CD8 T cell-mediated tumor rejection. In addition, secondary tumor challenge at a distal site was completely suppressed in mice treated with a dual-module adenovirus. These results suggest that a dual-targeting strategy to elicit both tumor antigen priming and tumor-induced immunoresistance enhances CD8 T cell-mediated antitumor immunity.
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Antígeno B7-H1/genética , Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/uso terapéutico , Vectores Genéticos , Timidina Quinasa/genética , Adenoviridae/genética , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/inmunología , Antineoplásicos/farmacología , Antígeno B7-H1/metabolismo , Vacunas contra el Cáncer/inmunología , Línea Celular Tumoral , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Femenino , Inmunidad Celular/genética , Inmunidad Celular/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Linfocitos T Citotóxicos/inmunología , Telomerasa/genética , Telomerasa/metabolismo , Timidina Quinasa/metabolismo , Trans-EmpalmeRESUMEN
Recombinant adenovirus (rAdV) vector is the most promising vehicle to deliver an exogenous gene into target cells and is preferred for gene therapy. Exogenous gene expression from rAdV is often too inefficient to induce phenotypic changes and the amount of administered rAdV must be very high to achieve a therapeutic dose. However, it is often hampered because a high dose of rAdV is likely to induce cytotoxicity by activating immune responses. nc886, a 102-nucleotide non-coding RNA that is transcribed by RNA polymerase III, acts as an immune suppressor and a facilitator of AdV entry into the nucleus. Therefore, in this study, we have constructed an rAdV expressing nc886 (AdV:nc886) to explore whether AdV:nc886 overcomes the aforementioned drawbacks of conventional rAdV vectors. When infected into mouse cell lines and mice, AdV:nc886 expresses a sufficient amount of nc886, which suppresses the induction of interferon-stimulated genes and apoptotic pathways triggered by AdV infection. As a result, AdV:nc886 is less cytotoxic and produces more rAdV-delivered gene products, compared with the parental rAdV vector lacking nc886. In conclusion, this study demonstrates that the nc886-expressing rAdV could become a superior gene delivery vehicle with greater safety and higher efficiency for in vivo gene therapy.
RESUMEN
Microtubule inhibitors (MTIs) such as Taxol have been used for treating various malignant tumors. Although MTIs have been known to induce cell death through mitotic arrest, other mechanisms can operate in MTI-induced cell death. Especially, the role of p53 in this process has been controversial for a long time. Here we investigated the function of p53 in Taxol-induced apoptosis using p53 wild type and p53 null cancer cell lines. p53 was upregulated upon Taxol treatment in p53 wild type cells and deletion of p53 diminished Taxol-induced apoptosis. p53 target proteins including MDM2, p21, BAX, and ß-isoform of PUMA were also upregulated by Taxol in p53 wild type cells. Conversely, when the wild type p53 was re-introduced into two different p53 null cancer cell lines, Taxol-induced apoptosis was enhanced. Among post-translational modifications that affect p53 stability and function, p53 acetylation, rather than phosphorylation, increased significantly in Taxol-treated cells. When acetylation was enhanced by anti-Sirt1 siRNA or an HDAC inhibitor, Taxol-induced apoptosis was enhanced, which was not observed in p53 null cells. When an acetylation-defective mutant of p53 was re-introduced to p53 null cells, apoptosis was partially reduced compared to the re-introduction of the wild type p53. Thus, p53 plays a pro-apoptotic role in Taxol-induced apoptosis and acetylation of p53 contributes to this pro-apoptotic function in response to Taxol in several human cancer cell lines, suggesting that enhancing acetylation of p53 could have potential implication for increasing the sensitivity of cancer cells to Taxol.
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Apoptosis/efectos de los fármacos , Paclitaxel/farmacología , Proteína p53 Supresora de Tumor/metabolismo , Acetilación , Línea Celular Tumoral , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
UNLABELLED: FX is an RNA-Seq analysis tool, which runs in parallel on cloud computing infrastructure, for the estimation of gene expression levels and genomic variant calling. In the mapping of short RNA-Seq reads, FX uses a transcriptome-based reference primarily, generated from ~160 000 mRNA sequences from RefSeq, UCSC and Ensembl databases. This approach reduces the misalignment of reads originating from splicing junctions. Unmapped reads not aligned on known transcripts are then mapped on the human genome reference. FX allows analysis of RNA-Seq data on cloud computing infrastructures, supporting access through a user-friendly web interface. AVAILABILITY: FX is freely available on the web at (http://fx.gmi.ac.kr), and can be installed on local Hadoop clusters. Guidance for the installation and operation of FX can be found under the 'Documentation' menu on the website. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
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Perfilación de la Expresión Génica , Análisis de Secuencia de ARN , Interfaz Usuario-Computador , Genoma , Genoma Humano , Humanos , Sitios de Empalme de ARN , Empalme del ARN , ARN Mensajero/genéticaRESUMEN
In an effort to identify novel genes related to the prognosis of gastric cancer, we performed gene expression profiling and found overexpressed levels of human interferon-induced transmembrane protein 1 (IFITM1). We validated the gastric cancer-specific up-regulation of IFITM1 and its association with cancer progression. We also studied its epigenetic regulation and tumorigenesis-related functions. Expression of IFITM1 was evaluated in various human gastric cancer cells and in 35 patient tumor tissues by quantitative RT-PCR and Western blot analyses. The results showed highly up-regulated IFITM1 in cancer cell lines and tissues. Furthermore, IHC studies were performed on 151 patient tissues, and a significant correlation was revealed between higher IFITM1 expression and Lauren's intestinal type (P = 0.007) and differentiated adenocarcinoma (P = 0.025). Quantitative studies of DNA methylation for 27 CpG sites in the regulatory region showed hypermethylation in cells expressing low levels of IFITM1. Methylation-dependent IFITM1 expression was confirmed further by in vitro demethylation using 5-aza-2'-deoxycytidine and luciferase assays. The functional analysis of IFITM1 by silencing of its expression with small-interfering RNA showed decreased migration and invasiveness of cancer cells, whereas its overexpression exhibited the opposite results. In this study, we demonstrated gastric cancer-specific overexpression of IFITM1 regulated by promoter methylation and the role of IFITM1 in cancer prognosis.
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Antígenos de Diferenciación/biosíntesis , Biomarcadores de Tumor/biosíntesis , Epigénesis Genética/fisiología , Regulación Neoplásica de la Expresión Génica/fisiología , Neoplasias Gástricas/genética , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anciano , Antígenos de Diferenciación/genética , Biomarcadores de Tumor/genética , Movimiento Celular/fisiología , Islas de CpG/genética , Metilación de ADN , ADN de Neoplasias/genética , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Masculino , Persona de Mediana Edad , Invasividad Neoplásica , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Estadificación de Neoplasias , Pronóstico , ARN Mensajero/genética , ARN Neoplásico/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Células Tumorales Cultivadas , Regulación hacia Arriba/fisiologíaRESUMEN
Owing to a lack of reliable markers and therapeutic targets, pancreatic ductal adenocarcinoma (PDAC) remains the most lethal malignant tumor despite numerous therapeutic advances. In this study, we utilized cell-SELEX to isolate a DNA aptamer recognizing the natural conformation of the target on the cell surface. PAp7T8, an aptamer optimized by size and chemical modification, exhibited specific targeting to pancreatic cancer cells and orthotopic xenograft pancreatic tumors. To confer therapeutic functions to the aptamer, we adopted a drug-conjugated oligobody (DOligobody) strategy. Monomethyl auristatin E was used as a cytotoxic drug, digoxigenin acted as a hapten, and the humanized anti-digoxigenin antibody served as a universal carrier of the aptamer. The resulting PAp7T8-DOligobody showed extended in vivo half-life and markedly inhibited tumor growth in an orthotopic pancreatic cancer xenograft model without causing significant toxicity. Therefore, PAp7T8-DOligobody represents a promising novel therapeutic delivery platform for PDAC.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Preparaciones Farmacéuticas , Línea Celular Tumoral , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/patología , Anticuerpos , Oligonucleótidos/uso terapéutico , Ensayos Antitumor por Modelo de Xenoinjerto , Neoplasias PancreáticasRESUMEN
Rotenone exposure has emerged as an environmental risk factor for inflammation-associated neurodegenerative diseases. However, the underlying mechanisms responsible for the harmful effects of rotenone in the brain remain poorly understood. Herein, we report that myeloperoxidase (MPO) may have a potential regulatory role in rotenone-exposed brain-resident immune cells. We show that microglia, unlike neurons, do not undergo death; instead, they exhibit distinctive activated properties under rotenone-exposed conditions. Once activated by rotenone, microglia show increased production of reactive oxygen species, particularly HOCl. Notably, MPO, an HOCl-producing enzyme that is undetectable under normal conditions, is significantly increased after exposure to rotenone. MPO-exposed glial cells also display characteristics of activated cells, producing proinflammatory cytokines and increasing their phagocytic activity. Interestingly, our studies with MPO inhibitors and MPO-knockout mice reveal that MPO deficiency potentiates, rather than inhibits, the rotenone-induced activated state of glia and promotes glial cell death. Furthermore, rotenone-triggered neuronal injury was more apparent in co-cultures with glial cells from Mpo(-/-) mice than in those from wild-type mice. Collectively, our data provide evidence that MPO has dual functionality under rotenone-exposed conditions, playing a critical regulatory role in modulating pathological and protective events in the brain.
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Encéfalo/metabolismo , Peroxidasa/fisiología , Rotenona/farmacología , Animales , Supervivencia Celular , Femenino , Humanos , Sistema Inmunológico , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Modelos Biológicos , Neuronas/metabolismo , Peroxidasa/genética , Fagocitosis , Ratas , Ratas Sprague-Dawley , Desacopladores/farmacologíaRESUMEN
Elucidation of the interplay between viruses and host cells is crucial for taming viruses to benefit human health. Cancer therapy using adenovirus, called oncolytic virotherapy, is a promising treatment option but is not robust in all patients. In addition, inefficient replication of human adenovirus in mouse hampered the development of an in vivo model for preclinical evaluation of therapeutically engineered adenovirus. nc886 is a human non-coding RNA that suppresses Protein Kinase R (PKR), an antiviral protein. In this study, we have found that nc886 greatly promotes adenoviral gene expression and replication. Remarkably, the stimulatory effect of nc886 is not dependent on its function to inhibit PKR. Rather, nc886 facilitates the nuclear entry of adenovirus via modulating the kinesin pathway. nc886 is not conserved in mouse and, when xenogeneically expressed in mouse cells, promotes adenovirus replication. Our investigation has discovered a novel mechanism of how a host ncRNA plays a pro-adenoviral role. Given that nc886 expression is silenced in a subset of cancer cells, our study highlights that oncolytic virotherapy might be inefficient in those cells. Furthermore, our findings open future possibilities of harnessing nc886 to improve the efficacy of oncolytic adenovirus and to construct nc886-expressing transgenic mice as an animal model.
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Human cytoskeleton-associated protein 2 (hCKAP2) is upregulated and highly expressed in various human malignances. hCKAP2 has microtubule-stabilizing characteristics and potentially regulates the dynamics and assembly of the mitotic spindle and chromosome segregation, indicating that hCKAP2 plays important functions during mitosis. In this study, we evaluated hCKAP2 as a plausible anticancer target through development and validation of a targeted cancer gene therapy strategy based on targeting and replacement of hCKAP2 RNA using a trans-splicing ribozyme. This targeted RNA replacement triggered transgene activity via accurate trans-splicing reaction selectively in human cancer cells expressing the hCKAP2 RNA and simultaneously reduced the expression level of the RNA in the cells. Adenoviral vector encoding the hCKAP2-specific trans-splicing ribozyme selectively induced cytotoxicity in tumor cells expressing hCKAP2. Moreover, intratumoral injection of the virus produced selective and efficient regression of tumor that had been subcutaneously inoculated with hCKAP2-positive colon cancer cells in mice with minimal liver toxicity. Furthermore, orthotopically multifocal hCKAP2-positive hepatocarcinoma established in mice were efficiently regressed by systemic delivery of adenoviral vector encoding the specific ribozyme under the control of a liver-selective phosphoenolpyruvate carboxykinase promoter with least hepatotoxicity. The results indicate that hCKAP2 RNA is a promising target for anticancer approach based on trans-splicing ribozyme-mediated RNA replacement.
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Proteínas del Citoesqueleto/genética , Terapia Genética , Neoplasias/genética , Neoplasias/terapia , ARN Catalítico , Trans-Empalme , Transgenes/fisiología , Animales , Proliferación Celular , Células Cultivadas , Enfermedad Hepática Inducida por Sustancias y Drogas/prevención & control , Vectores Genéticos/uso terapéutico , Humanos , Inyecciones Intralesiones , Masculino , Ratones , Ratones Endogámicos BALB C , Fosfoenolpiruvato Carboxilasa/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Gastric cancer remains the fourth most prevalent cancer and the second leading cause of cancer-related death in the world. The predominant form of gastric cancer is adenocarcinoma, which originates from glandular epithelium of the gastric mucosa. The major risk factors for gastric cancer include diet, individual genetic variation, and, most importantly, infection with Helicobacter pylori (H. pylori). Certain strains of H. pylori assisted by some of its virulence factors seem to play a critical role in gastric cancer development. Several of these H. pylori virulence factors, which influence cellular proliferation signaling, have been identified. In addition, changes in the expression of several cell proliferation regulating genes accompany or cause the progression of gastric cancer. These changes include modifications of cell cycle regulators, oncogene activation, tumor suppressor inactivation, and miRNA profile alterations. Many of these changes result from H. pylori infection, although their impact on the cellular proliferation system underlying gastric cancer development has not yet been fully elucidated. We review certain features of gastric cancer, the role of H. pylori infection in its etiology and pathogenesis, and gene expression changes during gastric carcinogenesis.
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Adenocarcinoma/patología , Proliferación Celular , Neoplasias Gástricas/patología , Adenocarcinoma/genética , Adenocarcinoma/microbiología , Ciclo Celular , Mucosa Gástrica/patología , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/patogenicidad , Humanos , Transducción de Señal , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiología , Factores de Virulencia/metabolismoRESUMEN
BACKGROUND: Group I intron-based trans-splicing ribozyme, which can specifically reprogram human telomerase reverse transcriptase (hTERT) RNA, could be a useful tool for tumor-targeted gene therapy. In the present study, the therapeutic feasibility of this ribozyme was investigated by analyzing trans-splicing efficacy in vivo as well as in cells. METHODS: We assessed transgene activation, degree of ribozyme expression, targeted hTERT mRNA level, or the level of trans-splicing products in hTERT(+) cells or in human tumor nodules xenografted in animals after ribozyme administration. RESULTS: The activity and efficacy of the trans-splicing ribozyme in cells was dependent on the amount of endogenous hTERT mRNA and/or the accumulation of ribozyme RNA in cells. Intracellular activity of the ribozyme reached a plateau when no more targetable substrate mRNA was available or the ribozyme RNA level was fully saturated. In addition, the efficacy of ribozyme in xenografted tumor tissues was dependent on the dose of the delivered ribozyme-encoding adenoviral vector, indicating the potential of the ribozyme expression level as a determining factor for the in vivo efficacy of the trans-splicing ribozyme. On the basis of these results, we enhanced the intracellular ribozyme activity by increasing the ribozyme expression level transcriptionally and/or post-transcriptionally. CONCLUSIONS: We analyzed ribozyme efficacy and determined the most influential factors of its trans-splicing reaction in mammalian cell lines as well as in vivo. The present study could provide insights into the optimization of the trans-splicing ribozyme-based RNA replacement approach to cancer treatment.
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Espacio Intracelular/enzimología , Intrones/genética , Neoplasias/enzimología , ARN Catalítico/genética , ARN Catalítico/metabolismo , Adenoviridae/genética , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Vectores Genéticos/genética , Células HEK293 , Células HT29 , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/genética , Neoplasias/patología , Telomerasa/metabolismo , Trasplante HeterólogoRESUMEN
TCR stimulation not only initiates positive signals for T cell activation, but also induces negative signals that down-regulate T cells. We previously reported that Sprouty1, a negative regulator of Ras-MAPK pathway of receptor tyrosine kinases, was induced by TCR signal and inhibited TCR signaling in CD4+ T cell clones. In this study, we addressed the mechanism underlying Sprouty1 inhibition of T cells. When overexpressed in Jurkat T cells, Sprouty1 inhibited TCR signal-induced IL-2 transcription, and also AP-1, NFAT, and NF-kappaB activation, which suggests that Sprouty1 acts at proximal TCR signalosome. Accordingly, we found that Sprouty1 translocated to immune synapse upon TCR engagement in both Jurkat cells and activated primary T cells and interacted with various signaling molecules in the TCR signalosome, such as linker for activation of T cells (LAT), phospholipase C-gamma1 (PLC-gamma1), c-Cbl/Cbl-b, and HPK1. Sprouty1 inhibited LAT phosphorylation, leading to decreased MAPK activation and IL-2 production. Deletion of C-terminal 54 amino acids in Sprouty1 abolished its inhibitory effect and this deletion mutant was unable to translocate to immune synapse and interact with LAT. Overall, our data suggest that Sprouty1 induced by TCR signal negatively regulates further TCR signaling by interacting with proximal signaling molecules in immune synapse, providing a novel regulatory mechanism of T cells.