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Toca 511, a tumor-selective retroviral replicating vector encoding the yeast cytosine deaminase (yCD) gene, exerts direct antitumor effects through intratumoral prodrug 5-fluorocytosine (5-FC) conversion to active drug 5-fluorouracil by yCD, and has demonstrated therapeutic efficacy in preclinical and clinical trials of various cancers. Toca 511/5-FC treatment may also induce antitumor immunity. Here, we first examined antitumor immune responses activated by Toca 511/5-FC treatment in an immunocompetent murine pancreatic cancer model. We then evaluated the therapeutic effects achieved in combination with anti-programmed cell death protein 1 antibody. In the bilateral subcutaneous tumor model, as compared with the control group, enhanced CD8+ T-cell-mediated cytotoxicity and increased T-cell infiltration in Toca 511-untransduced contralateral tumors were observed. Furthermore, the expression levels of T-cell co-inhibitory receptors on CD8+ T-cells increased during treatment. In the bilateral subcutaneous tumor model, combination therapy showed significantly stronger tumor growth inhibition than that achieved with either monotherapy. In an orthotopic tumor and peritoneal dissemination model, the combination therapy resulted in complete regression in both transduced orthotopic tumors and untransduced peritoneal dissemination. Thus, Toca 511/5-FC treatment induced a systemic antitumor immune response, and the combination therapy could be a promising clinical strategy for treating metastatic pancreatic cancer.
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Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated impressive success in the treatment of patients with hematologic tumors yet achieved very limited efficacy for solid tumors due to hurdles unique to solid tumors. It is also noted that the tumor microenvironment composition varies between tumor type, which again imposes unique set of hurdles in each solid tumor. Therefore, elucidation of individual hurdles is key to achieving successful CAR-T therapy for solid tumors. In the present study, we employed an orthotopic human PDAC xenograft model, in which quantitative, spatial and functional dynamics of CAR-T cells in tumor tissues were analyzed to obtain insights into ways of overcoming PDAC related hurdles. Contrary to previous studies that demonstrated a limited persistency and infiltration of CAR-T cells in many solid tumors, they persist and accumulated in PDAC tumor tissues. Ex vivo analysis revealed that CAR-T cells that had been recovered at different time points from mice bearing an orthotopic PDAC tumor exhibited a gradual loss of tumor reactivity. This loss of tumor reactivity of CAR-T cells was associated with the increased expression of AMP-activated protein kinase and Mitofusin 1/ Dynamin-related protein 1 ratio.
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Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Animales , Ratones , Receptores de Antígenos de Linfocitos T , Linfocitos T , Xenoinjertos , Inmunoterapia Adoptiva , Neoplasias/metabolismo , Microambiente TumoralRESUMEN
The epithelial-mesenchymal transition (EMT) contributes to the metastatic cascade in various tumors. C-C chemokine receptor 7 (CCR7) interacts with its ligand, chemokine (C-C motif) ligand 19 (CCL19), to promote EMT. However, the association between EMT and CCR7 in extrahepatic cholangiocarcinoma (EHCC) remains unknown. This study aimed to elucidate the prognostic impact of CCR7 expression and its association with clinicopathological features and EMT in EHCC. The association between CCR7 expression and clinicopathological features and EMT status was examined via the immunohistochemical staining of tumor sections from 181 patients with perihilar cholangiocarcinoma. This association was then investigated in TFK-1 and EGI-1 EHCC cell lines. High-grade CCR7 expression was significantly associated with a large number of tumor buds, low E-cadherin expression, and poor overall survival. TFK-1 showed CCR7 expression, and Western blotting revealed E-cadherin downregulation and vimentin upregulation in response to CCL19 treatment. The wound healing and Transwell invasion assays revealed that the activation of CCR7 by CCL19 enhanced the migration and invasion of TFK-1 cells, which were abrogated by a CCR7 antagonist. These results suggest that a high CCR7 expression is associated with an adverse postoperative prognosis via EMT induction and that CCR7 may be a potential target for adjuvant therapy in EHCC.
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Chimeric antigen receptor engineered T cell (CAR-T) therapy has high therapeutic efficacy against blood cancers, but it has not shown satisfactory results in solid tumors. Therefore, we examined the therapeutic effect of CAR-T therapy targeting carcinoembryonic antigen (CEA) in pancreatic adenocarcinoma (PDAC). CEA expression levels on the cell membranes of various PDAC cell lines were evaluated using flow cytometry and the cells were divided into high, medium, and low expression groups. The relationship between CEA expression level and the antitumor effect of anti-CEA-CAR-T was evaluated using a functional assay for various PDAC cell lines; a significant correlation was observed between CEA expression level and the antitumor effect. We created orthotopic PDAC xenograft mouse models and injected with anti-CEA-CAR-T; only the cell line with high CEA expression exhibited a significant therapeutic effect. Thus, the therapeutic effect of CAR-T therapy was related to the target antigen expression level, and the further retrospective analysis of pathological findings from PDAC patients showed a correlation between the intensity of CEA immunostaining and tumor heterogeneity. Therefore, CEA expression levels in biopsies or surgical specimens can be clinically used as biomarkers to select PDAC patients for anti-CAR-T therapy.
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Therapeutic efficacy of retroviral replicating vector (RRV)-mediated prodrug activator gene therapy has been demonstrated in a variety of tumor models, but clinical investigation of this approach has so far been restricted to glioma and gastrointestinal malignancies. In the present study, we evaluated replication kinetics, transduction efficiency, and therapeutic efficacy of RRV in experimental models of lung cancer. RRV delivering GFP as a reporter gene showed rapid viral replication in a panel of lung cancer cells in vitro, as well as robust intratumoral replication and high levels of tumor transduction in subcutaneous and orthotopic pleural dissemination models of lung cancer in vivo. Toca 511 (vocimagene amiretrorepvec), a clinical-stage RRV encoding optimized yeast cytosine deaminase (yCD) which converts the prodrug 5-fluorocytosine (5-FC) to the active drug 5-fluorouracil (5-FU), showed potent cytotoxicity in lung cancer cells upon exposure to 5-FC prodrug. In vivo, Toca 511 achieved significant tumor growth inhibition following 5-FC treatment in subcutaneous and orthotopic pleural dissemination models of lung cancer in both immunodeficient and immunocompetent hosts, resulting in significantly increased overall survival. This study demonstrates that RRV can serve as highly efficient vehicles for gene delivery to lung cancer, and indicates the translational potential of RRV-mediated prodrug activator gene therapy with Toca 511/5-FC as a novel therapeutic strategy for pulmonary malignancies.
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Daikenchuto (DKT) is a Japanese traditional herbal (Kampo) medicine containing ginseng, processed ginger, and Japanese or Chinese pepper. We aimed to determine how DKT affects human colonic microbiota. An in vitro microbiota model was established using fecal inocula collected from nine healthy volunteers, and each model was found to retain operational taxonomic units similar to the ones in the original human fecal samples. DKT was added to the in vitro microbiota model culture at a concentration of 0.5% by weight. Next-generation sequencing of bacterial 16S rRNA gene revealed a significant increase in the relative abundance of bacteria related to the Bifidobacterium genus in the model after incubation with DKT. In pure cultures, DKT significantly promoted the growth of Bifidobacterium adolescentis, but not that of Fusobacterium nucleatum or Escherichia coli. Additionally, in pure cultures, B. adolescentis transformed ginsenoside Rc to Rd, which was then probably utilized for its growth. Our study reveals the in vitro bifidogenic effect of DKT that likely contributes to its beneficial effects on the human colon.
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Bifidobacterium/efectos de los fármacos , Colon/microbiología , Microbioma Gastrointestinal , Extractos Vegetales/farmacología , Bifidobacterium/crecimiento & desarrollo , Bifidobacterium/aislamiento & purificación , Trasplante de Microbiota Fecal , Microbioma Gastrointestinal/genética , Humanos , Técnicas In Vitro , Panax , ARN Ribosómico 16S/genética , Análisis de Secuencia de ARN/métodos , Zanthoxylum , ZingiberaceaeRESUMEN
The intestine acts as a center for nutrient and water absorption at the epithelium and plays an important role in immunity. Considering the complexity of its function and roles in living systems, a physiologically relevant gut in vitro model is desirable in both basic biology and the analysis of effects of some substances on functions of the gut; these analyses include the screening of drug and food candidates with regard to intestinal disorder at an early stage of medical development. In the present study, we constructed a three-dimensional (3D) gut model using human absorptive enterocytes (CACO-2 cells) by reconstitution of the gut epithelial sheet restricted on a high-reproducible ductal scaffold of collagen gel. Moreover, using the 3D gut model, we evaluated the morphology at the cellular and tissue levels and conducted a phenotypic analysis of the intestinal physiological functions, which involved a permeability assay mimicking barrier disruption inducing inflammation and an absorption assay reflecting ingestive effects. The ductal structure, in vivo-like 3D epithelial structures, epithelial barrier, and effective absorptive function characterized the 3D gut model. The epithelial cells formed a villus-like buckling epithelium, vertical microvilli of increased density on the cell surface, and a crypt-like localized cell proliferating region. The mature shape of the epithelium may contribute to mimicking barrier function and effective absorption compared with that in the 2D gut model. Furthermore, we successfully mimicked the dextran sodium sulfate-induced epithelial barrier dysfunction as a trigger phenomenon of gut inflammation in the 3D gut model. The integrity of the epithelium and phenotypic analysis of the intestinal physiological functions in the simple and reproducible 3D gut model will allow for a drug screening system for assessing the effects on the functions of the gut epithelium from the lumen side.
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Microbioma Gastrointestinal , Células CACO-2 , Células Epiteliales , Humanos , Mucosa Intestinal , IntestinosRESUMEN
Over the past 30 years, the therapeutic outcome for pancreatic ductal adenocarcinoma (PDAC) has remained stagnant due to the lack of effective treatments. We performed a genome-wide analysis to identify novel therapeutic targets for PDAC. Our analysis showed that Homo sapiens chromosome 16 open reading frame 74 (C16orf74) was upregulated in most patients with PDAC and associated with poor prognosis. Previously, we demonstrated that C16orf74 interacts with the catalytic subunit alpha of protein phosphatase 3 and plays an important role in PDAC invasion. However, the pathophysiologic function of C16orf74 is still unclear. In this study, through the analysis of C16orf74 interaction, we demonstrate a new strategy to inhibit the growth and invasion of PDAC. C16orf74 exists in the homodimer form under the cell membrane and binds integrin αVß3 and is also involved in invasion by activating Rho family (Rac1) and MMP2. Considering that this dimeric form was found to be involved in the function of C16orf74, we designed an 11R-DB (dimer block) cell-permeable dominant-negative peptide that inhibits the dimer form of C16orf74. 11R-DB suppressed invasion and proliferation of PDAC cell lines by inhibiting phosphorylation of Akt and mTOR and also by inactivation of MMP2. 11R-DB also showed antitumor effects in an orthotopic xenograft model and peritoneal metastasis model. Thus, this study demonstrates that dimerized C16orf74, present in the cell membrane, is involved in pancreatic cancer invasion and proliferation. In addition, the C16orf74 dimer block cell-permeable peptide (11R-DB) has a potent therapeutic effect on PDAC in vitro and in vivo.
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Biomarcadores de Tumor/genética , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Animales , Biomarcadores de Tumor/metabolismo , Células COS , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , Chlorocebus aethiops , Femenino , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Integrina alfaVbeta3/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Terapia Molecular Dirigida , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Péptidos/farmacología , Multimerización de Proteína , Transducción de Señal , Regulación hacia Arriba , Neoplasias PancreáticasRESUMEN
We established mutated and non-mutated induced pluripotent stem cell (iPSC) clones from a patient with PTPN11 (c.226G>A)-mutated juvenile myelomonocytic leukaemia (JMML). Both types of iPSCs fulfilled the quality criteria. Mutated iPSC colonies generated significantly more CD34+ and CD34+ CD45+ cells compared to non-mutated iPSC colonies in a culture coated with irradiated AGM-S3 cells to which four growth factors were added sequentially or simultaneously. The haematopoietic differentiation potential of non-mutated JMML iPSC colonies was similar to or lower than that of iPSC colonies from a healthy individual. The PTPN11 mutation coexisted with the OSBP2 c.389C>T mutation. Zinc-finger nuclease-mediated homologous recombination revealed that correction of PTPN11 mutation in iPSCs with PTPN11 and OSBP2 mutations resulted in reduced CD34+ cell generation to a level similar to that obtained with JMML iPSC colonies with the wild-type of both genes, and interestingly, to that obtained with normal iPSC colonies. Transduction of the PTPN11 mutation into JMML iPSCs with the wild-type of both genes increased CD34+ cell production to a level comparable to that obtained with JMML iPSC colonies harbouring the two genetic mutations. Thus, PTPN11 mutation may be the most essential abnormality to confer an aberrant haematopoietic differentiation potential in this disorder.
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Diferenciación Celular/genética , Células Madre Hematopoyéticas/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Leucemia Mielomonocítica Juvenil , Células Madre Neoplásicas/metabolismo , Mutación Puntual , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Animales , Células Madre Hematopoyéticas/patología , Humanos , Células Madre Pluripotentes Inducidas/patología , Leucemia Mielomonocítica Juvenil/genética , Leucemia Mielomonocítica Juvenil/metabolismo , Leucemia Mielomonocítica Juvenil/patología , Masculino , Ratones SCID , Células Madre Neoplásicas/patología , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismoRESUMEN
It has been clear that the clinical responses by applying immune checkpoint inhibitor alone are limited. To better improve this limited clinical response, combinational therapy has been focused. We recently reported the association between EMT related factors and PD-L1 expression in the extrahepatic hilar cholangiocarcinoma, and its role as a surrogate biomarker for patient prognosis. We here report clinical relevance of combinational therapy of HDAC inhibitors and anti-PD-L1 antibody as an immune checkpoint inhibitors.
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Microambiente Tumoral , Antígeno B7-H1 , Neoplasias de los Conductos Biliares , Conductos Biliares Intrahepáticos , Transición Epitelial-Mesenquimal , Humanos , Receptor de Muerte Celular Programada 1RESUMEN
The efficacy of preoperative neoadjuvant chemoradiotherapy (NAC) in cases of pancreatic cancer with extremely poor prognoses has been reported. In this study, we aimed to identify novel biomarkers that reflect prognoses following chemoradiotherapy using tertiary lymphoid organs (TLO) expressed in the tumor microenvironment. Resected tumor specimens were obtained from 140 pancreatic cancer patients. We retrospectively investigated the clinical relevance of TLO by categorizing patients into those who underwent upfront surgery (surgery first [SF]) and those who received NAC. The immunological elements within TLO were analyzed by immunohistochemistry (IHC). In the IHC analysis, the proportions of CD8+ T lymphocytes, PNAd+ high endothelial venules, CD163+ macrophages and Ki-67+ cells within the TLO were higher in the NAC group than in the SF group. In contrast, the proportion of programmed cell death-1+ immunosuppressive lymphocytes within TLO was lower in the NAC group than in the SF group. The NAC group demonstrated favorable prognoses compared with the SF group. In the multivariate analysis, the TLO/tumor ratio was determined as an independent predictive prognostic factor. In conclusion, the administration of preoperative chemoradiotherapy may influence the immunological elements in the tumor microenvironment and result in favorable prognoses in pancreatic ductal adenocarcinoma patients.
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Carcinoma Ductal Pancreático/inmunología , Tejido Linfoide/inmunología , Neoplasias Pancreáticas/inmunología , Microambiente Tumoral/inmunología , Biomarcadores de Tumor/inmunología , Biomarcadores de Tumor/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Carcinoma Ductal Pancreático/cirugía , Carcinoma Ductal Pancreático/terapia , Quimioradioterapia , Femenino , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Tejido Linfoide/metabolismo , Masculino , Persona de Mediana Edad , Terapia Neoadyuvante , Neoplasias Pancreáticas/cirugía , Neoplasias Pancreáticas/terapia , Pronóstico , Estudios RetrospectivosRESUMEN
Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. In this study, we firstly revealed that a new alternative HAMP transcript was found in hepatoma-derived cell line HLF, which was identical to the wild-type preprohepcidin sequence except lacking of an internal 60 bases. In addition to HLF, most of hepatoma-derived cell lines have significant copy numbers of variant-type hepcidin mRNA by a copy-based-digital PCR. Furthermore, the copy number of hepcidin mRNA variant was significantly higher in serum exosomes of hepatocellular carcinoma patients. The quantification of exosomal hepcidin mRNA variant may serve as a potential new biomarker for HCC diagnosis.
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Empalme Alternativo , Exosomas/metabolismo , Hepcidinas/metabolismo , Hierro/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Hepatocelular/metabolismo , Estudios de Casos y Controles , Femenino , Variación Genética , Hepcidinas/genética , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Cancer stem cells (CSCs) are specific targets for therapeutic applications, but the rarity of CSCs within tumors makes the isolation of CSCs difficult. To overcome these problems, we generated CSCs in vitro using established reprogramming techniques. We transduced four previously established reprogramming factors, Oct3/4, Sox2, Klf4, and L-myc, into the colon cancer cell lines LoVo and OUMS-23, and investigated the biological characteristics of these lines. Tra-1-60+ cells were obtained from reprogrammed induced pluripotent stem (iPS) cell-like colonies and showed CSC properties, including colony formation, maintenance of colonies by repeated passages, and feeder cell dependency, as well as increased expressions of CSC markers such as CD133 and ALDH1. The CSC-like cells showed increased chemoresistance to 5-fluorouracil and elevated tumorigenicity upon transplantation into kidneys of immune-deficient mice. These tumors shifted to a poorly differentiated stage with many atypical cells, cytoplasmic mucin, and focal papillary components, with demonstrated dedifferentiation. The principal component analysis from DNA microarrays showed that though both cell lines moved to iPS cells after reprogramming, they were not completely identical to iPS cells. Significantly elevated gene expression of Decorin and CD90 was observed in CSC-like cells. Together, these results show that reprogramming of cancer cells produced not pluripotent stem cells but CSC-like cells, and these findings will provide biological information about genuine CSCs and help establish new CSC-targeted therapies.
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Reprogramación Celular , Neoplasias del Colon/patología , Células Madre Neoplásicas/metabolismo , Animales , Biomarcadores de Tumor/metabolismo , Células Madre Pluripotentes Inducidas/citología , Factor 4 Similar a Kruppel , Ratones SCID , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The gastric cardia-the small area around the cardiac orifice including the abdominal esophagus-is an important target area for abdominal and thoracic surgeries, especially for laparoscopic procedures. In this study of 28 cadavers, a peritoneal earlobe-like appendage near the angle of His was identified as a useful indicator of the lateral margin of the abdominal esophagus, which is otherwise obscure because the peritoneum continues to the diaphragm without definite demarcation of this margin. This structure, which appears equivalent to the epiploic appendages, was commonly found to be present (in 22/28, 78.6 % of the 28 cadavers) and was 4-21 mm × 6-40 mm × 1-4 mm in size, triangular, round, or leaf-like in shape, contained fat, and was on an imaginary line along which the lesser omentum adheres to the lesser curvature and continues to the diaphragm (18/22, 81.8 %). This indicator is associated with the lesser omentum and is part of the gastrophrenic ligament, and could serve as a useful indicator of the margin of the gastric cardia, thus aiding surgeons performing laparoscopic surgery in this region.
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Cardias/anatomía & histología , Anciano , Anciano de 80 o más Años , Diafragma/anatomía & histología , Esófago/anatomía & histología , Femenino , Humanos , Laparoscopía , Masculino , Márgenes de Escisión , Persona de Mediana Edad , Epiplón/anatomía & histología , Peritoneo/anatomía & histologíaRESUMEN
Understanding the biological and molecular processes underlying human pathologies is fundamental in order to develop innovative approaches to treat or prevent them. Among the technologies that could provide innovative disease models, induced pluripotent stem cells (iPSCs) is one of the most promising. Indeed, one application of this technology is patient-specific disease modeling. iPSCs obtained by reprogramming patients' cells collected from accessible tissues, have the unique capability to differentiate, under an adequate stimulus, into any human cell type. In particular, iPSCs technology can be applied to study drug adverse effects, that is a key part of the drug discovery process. Indeed, drug induced adverse effects are among the most common causes that lead to abandon the development of new candidate therapeutic molecules, increasing the cost of drug discovery. An innovative strategy that could be used in drug design to solve drug attrition rate, and to establish innovative pharmacological models, could be the application of iPSCs technology in the early stage of the drug discovery process to model druginduced adverse events. In this review, recently developed disease models based on iPSCs will be discussed, with a particular focus on available models of drugs' adverse effect, in particular hepatic/pancreatic toxicity.
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Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Reprogramación Celular , Niño , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos/etiología , Enfermedad Granulomatosa Crónica/tratamiento farmacológico , Enfermedad Granulomatosa Crónica/patología , Humanos , Células Madre Pluripotentes Inducidas/citología , Hígado/efectos de los fármacos , Hígado/metabolismo , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Inhibidores de Proteínas Quinasas/uso terapéutico , Inhibidores de Proteínas Quinasas/toxicidadRESUMEN
Transfusion is believed to be the main cause of iron overload in Japan. A nationwide survey on post-transfusional iron overload subsequently led to the establishment of guidelines for iron chelation therapy in this country. To date, however, detailed clinical information on the entire iron overload population in Japan has not been fully investigated. In the present study, we obtained and studied detailed clinical information on the iron overload patient population in Japan. Of 1109 iron overload cases, 93.1% were considered to have occurred post-transfusion. There were, however, 76 cases of iron overload of unknown origin, which suggest that many clinicians in Japan may encounter some difficulty in correctly diagnosing and treating iron overload. Further clinical data were obtained for 32 cases of iron overload of unknown origin; median of serum ferritin was 1860.5 ng/mL. As occurs in post-transfusional iron overload, liver dysfunction was found to be as high as 95.7% when serum ferritin levels exceeded 1000 ng/mL in these patients. Gene mutation analysis of the iron metabolism-related genes in 27 cases of iron overload with unknown etiology revealed mutations in the gene coding hemojuvelin, transferrin receptor 2, and ferroportin; this indicates that although rare, hereditary hemochromatosis does occur in Japan.
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Sobrecarga de Hierro/epidemiología , Sobrecarga de Hierro/etiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Ferritinas/sangre , Hemocromatosis/diagnóstico , Hemocromatosis/epidemiología , Humanos , Hierro/metabolismo , Sobrecarga de Hierro/genética , Japón/epidemiología , Hepatopatías/etiología , Masculino , Redes y Vías Metabólicas/genética , Persona de Mediana Edad , Mutación , Encuestas y Cuestionarios , Reacción a la Transfusión , Adulto JovenRESUMEN
Retinoblastoma is the most common intraocular malignancy in pediatric patients. It develops rapidly in the retina and can be fatal if not treated promptly. It has been proposed that a small population of cancer cells, termed cancer stem cells (CSCs), initiate tumorigenesis from immature tissue stem cells or progenitor cells. Reprogramming technology, which can convert mature cells into pluripotent stem cells (iPS), provides the possibility of transducing malignant cancer cells back to CSCs, a type of early stage of cancer. We herein took advantage of reprogramming technology to induce CSCs from retinoblastoma cancer cells. In the present study, the 4 Yamanaka transcription factors, Oct4, Sox2, Klf4 and c-myc, were transduced into retinoblastoma cells (Rbc51). iPS-like colonies were observed 15 days after transduction and showed significantly enhanced CSC properties. The gene and protein expression levels of pluripotent stem cell markers (Tra-1-60, Oct4, Nanog) and cancer stem cell markers (CD133, CD44) were up-regulated in transduced Rbc51 cells compared to control cells. Moreover, iPS-like CSCs could be sorted using the Magnetic-activated cell sorting (MACS) method. A sphere formation assay demonstrated spheroid formation in transduced Rbc51 cells cultured in serum free media, and these spheroids could be differentiated into Pax6-, Nestin-positive neural progenitors and rhodopsin- and recoverin-positive mature retinal cells. The cell viability after 5-Fu exposure was higher in transduced Rbc51 cells. In conclusion, CSCs were generated from retinoblastoma cancer cells using reprogramming technology. Our novel method can generate CSCs, the study of which can lead to better understanding of cancer-specific initiation, cancer epigenetics, and the overlapping mechanisms of cancer development and pluripotent stem cell behavior.
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Técnicas de Reprogramación Celular/métodos , Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Células Madre Neoplásicas/citología , Neoplasias de la Retina/genética , Retinoblastoma/genética , Línea Celular Tumoral , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Factor 4 Similar a Kruppel , Células Madre Neoplásicas/metabolismo , Retina/citología , Retina/metabolismo , Factores de Transcripción/genética , Transducción GenéticaRESUMEN
Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are potential resources for the regeneration of defective organs, including the liver. However, some obstacles must be overcome before this becomes reality. Undifferentiated cells that remain following differentiation have teratoma-forming potential. Additionally, practical applications require a large quantity of differentiated cells, so the differentiation process must be economical. Here we describe a DNA microarray-based global analysis of the gene expression profiles of differentiating human pluripotent stem cells. We identified differences and commonalities among six human pluripotent stem cell lines: the hESCs KhES1, KhES2, KhES3, and H1, and the iPSCs 201B7 and 243G1. Embryoid bodies (EBs) formed without requiring supplementation with inducing factors. EBs also expressed some liver-specific metabolic genes including the ammonia-metabolizing enzymes glutamine synthetase and carbamoyl-phosphate synthase 1. Real-time PCR analysis revealed hepatocyte-like differentiation of EBs treated with ammonia in Lanford medium. Analysis of DNA microarray data suggested that hepatocyte-like cells were the most abundant population in ammonia-treated cells. Furthermore, expression levels of undifferentiated pluripotent stem cell markers were drastically reduced, suggesting a reduced teratoma-forming capacity. These results indicate that treatment of EBs with ammonia in Lanford medium may be an effective inducer of hepatic differentiation in absence of expensive inducing factors.
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Amoníaco/farmacología , Hepatocitos/efectos de los fármacos , Células Madre Pluripotentes/efectos de los fármacos , Animales , Diferenciación Celular , Linaje de la Célula , Hepatocitos/citología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Madre Pluripotentes/citologíaRESUMEN
Iron overload remains a concern in myelodysplastic syndrome (MDS) patients. Iron chelation therapy (ICT) thus plays an integral role in the management of these patients. Moreover, ICT has been shown to prolong leukemia-free survival in MDS patients; however, the mechanisms responsible for this effect are unclear. Iron is a key molecule for regulating cytosolic aconitase 1 (ACO1). Additionally, the mutation of isocitrate dehydrogenase (IDH), the enzyme downstream of ACO1 in the TCA cycle, is associated with epigenetic abnormalities secondary to 2-hydroxyglutarate (2-HG) and DNA methylation. However, epigenetic abnormalities observed in many MDS patients occur without IDH mutation. We hypothesized that iron itself activates the ACO1-IDH pathway, which may increase 2-HG and DNA methylation, and eventually contribute to leukemogenesis without IDH mutation. Using whole RNA sequencing of bone marrow cells in iron-overloaded mice, we observed that the enzymes, phosphoglucomutase 1, glycogen debranching enzyme, and isocitrate dehydrogenase 1 (Idh1), which are involved in glycogen and glucose metabolism, were increased. Digital PCR further showed that Idh1 and Aco1, enzymes involved in the TCA cycle, were also elevated. Additionally, enzymatic activities of TCA cycle and methylated DNA were increased. Iron chelation reversed these phenomena. In conclusion, iron activation of glucose metabolism causes an increase of 2-HG and DNA methylation.
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Médula Ósea/metabolismo , Metilación de ADN/efectos de los fármacos , Proteína 1 Reguladora de Hierro/metabolismo , Hierro/farmacología , Isocitrato Deshidrogenasa/metabolismo , Animales , Carcinogénesis/inducido químicamente , Glucosa/metabolismo , Glutaratos/sangre , Proteína 1 Reguladora de Hierro/efectos de los fármacos , Isocitrato Deshidrogenasa/efectos de los fármacos , RatonesRESUMEN
Hepcidin is a main regulator of iron metabolism, of which abnormal expression affects intestinal absorption and reticuloendothelial sequestration of iron by interacting with ferroportin. It is also noted that abnormal iron accumulation is one of the key factors to facilitate promotion and progression of cancer including hepatoma. By RT-PCR/agarose gel electrophoresis of hepcidin mRNA in a hepatocellular carcinoma cell line HLF, a smaller mRNA band was shown in addition to the wild-type hepcidin mRNA. From sequencing analysis, this additional band was a selective splicing variant of hepcidin mRNA lacking exon 2 of HAMP gene, producing the transcript that encodes truncated peptide lacking 20 amino acids at the middle of preprohepcidin. In the present study, we used the digital PCR, because such a small amount of variant mRNA was difficult to quantitate by the conventional RT-PCR amplification. Among seven hepatoma-derived cell lines, six cell lines have significant copy numbers of this variant mRNA, but not in one cell line. In the transient transfection analysis of variant-type hepcidin cDNA, truncated preprohepcidin has a different character comparing with native preprohepcidin: its product is insensitive to digestion, and secreted into the medium as a whole preprohepcidin form without maturation. Loss or reduction of function of HAMP gene by aberrantly splicing may be a suitable phenomenon to obtain the proliferating advantage of hepatoma cells.