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OBJECTIVE: Advanced cervical cancer is still difficult to treat and in the case of recurrent cancer, it is desirable to utilize personalized treatment rather than uniform treatment because the type of recurrence is different for each individual. Therefore, this study aimed to establish a patient-derived organoid (PDO) platform to determine the effects of chemotherapy, radiation therapy, and targeted therapy in cervical cancer. METHODS: We established organoids from 4 patients with various types of cervical cancer. The histopathological and gene profiles of these organoid models were compared to determine their characteristics and the maintenance of the patient phenotype. Each type of organoid was also subjected to anticancer drug screening and radiation therapy to evaluate its sensitivity. RESULTS: We established PDOs to recapitulate the main elements of the original patient tumors, including the DNA copy number and mutational profile. We selected 7 drugs that showed growth inhibition in cervical cancer organoids out of 171 using an Food and Drug Administration -approved drug library. Moreover, adenocarcinoma and large-cell neuroendocrine carcinoma showed resistance to radiation therapy. whereas squamous cell carcinoma and villoglandular carcinoma showed a significant response to radiotherapy. CONCLUSION: Our results showed that patient-derived cervical cancer organoids can be used as a platform for drug and radiation sensitivity testing. These findings suggest that patient-derived cervical cancer organoids could be used as a personalized medicine platform and may provide the best treatment options for patients with various subtypes of cervical cancer.
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Adenocarcinoma , Antineoplásicos , Carcinoma de Células Escamosas , Neoplasias del Cuello Uterino , Femenino , Humanos , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Medicina de Precisión , Neoplasias del Cuello Uterino/tratamiento farmacológico , Neoplasias del Cuello Uterino/genética , Recurrencia Local de Neoplasia/patología , Carcinoma de Células Escamosas/patología , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/genética , Organoides/patologíaRESUMEN
Stemness factors control microRNA expression in cancer stem cells. Downregulation of miR-100 and miR-125b is associated with tumor progression and prognosis of various cancers. Comparing miRNA profiling of patient-derived tumorsphere (TS) and adherent (2D) hepatocellular carcinoma cells, miR-100 and miR-125b are identified to have association with stemness. In TS cells, miR-100 and miR-125b were downregulated comparing to 2D cells. The finding was reproduced in Hep3B cells. Overexpression of stemness factors NANOG, OCT4 and SOX2 by introduction of gene constructs in Hep3B cells suppressed these two miRNA expression levels. Treatment of chromeceptin, an IGF signaling pathway inhibitor, decreased numbers of TS and inhibited the AKT/mTOR pathway. Stable cell line of miR-100 and miR-125b overexpression decreased IGF2 expression and inhibited tumor growth in the xenograft model. In conclusion, miR-100 and miR-125b have tumor suppressor role in hepatocellular carcinoma through inhibiting IGF2 expression and activation of the AKT/mTOR pathway.
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Carcinoma Hepatocelular/genética , Factor II del Crecimiento Similar a la Insulina/genética , Neoplasias Hepáticas/genética , MicroARNs/genética , Células Madre Neoplásicas/metabolismo , Regulación hacia Arriba , Benzopiranos/farmacología , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células Hep G2 , Humanos , Factor II del Crecimiento Similar a la Insulina/metabolismo , Neoplasias Hepáticas/metabolismo , Células Madre Neoplásicas/efectos de los fármacos , PronósticoRESUMEN
BACKGROUND: Development of chemotherapeutics for the treatment of advanced hepatocellular carcinoma (HCC) has been lagging. Screening of candidate therapeutic agents by using patient-derived preclinical models may facilitate drug discovery for HCC patients. METHODS: Four primary cultured HCC cells from surgically resected tumor tissues and six HCC cell lines were used for high-throughput screening of 252 drugs from the Prestwick Chemical Library. The efficacy and mechanisms of action of the candidate anti-cancer drug were analyzed via cell viability, cell cycle assays, and western blotting. RESULTS: Guanabenz acetate, which has been used as an antihypertensive drug, was screened as a candidate anti-cancer agent for HCC through a drug sensitivity assay by using the primary cultured HCC cells and HCC cell lines. Guanabenz acetate reduced HCC cell viability through apoptosis and autophagy. This occurred via inhibition of growth arrest and DNA damage-inducible protein 34, increased phosphorylation of eukaryotic initiation factor 2α, increased activating transcription factor 4, and cell cycle arrest. CONCLUSIONS: Guanabenz acetate induces endoplasmic reticulum stress-related cell death in HCC and may be repositioned as an anti-cancer therapeutic agent for HCC patients.
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Establishing an appropriate preclinical model is crucial for translational cancer research. The most common way that has been adopted by far is grafting cancer cell lines, derived from patients. Although this xenograft model is easy to generate, but has several limitations because this cancer model could not represent the unique features of each cancer patient sufficiently. Moreover, accumulating evidences demonstrate cancer is a highly heterogeneous disease so that a tumor is comprised of cancer cells with diverse characteristics. In attempt to avoid these discrepancies between xenograft model and patients' tumor, a patient-derived xenograft (PDX) model has been actively generated and applied. The PDX model can be developed by the implantation of cancerous tissue from a patient's tumor into an immune-deficient mouse directly, thereby it preserves both cell-cell interactions and tumor microenvironment. In addition, the PDX model has shown advantages as a preclinical model in drug screening, biomarker development and co-clinical trial. In this review, we will summarize the methodology and applications of PDX in detail, and cover critical issues for the development of this model for preclinical research.
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Neoplasias/terapia , Medicina de Precisión/métodos , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Neoplasias/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Although reverse transfection cell microarray (RTCM) is a powerful tool for mammalian cell studies, the technique is not appropriate for cells that are difficult to transfect. The lentivirus-infected cell microarray (LICM) technique was designed to improve overall efficiency. However, LICM presents new challenges because individual lentiviral particles can spread through the cell population, leading to cross-contamination. Therefore, we designed a cell-defined lentivirus microarray (CDLM) technique using cell-friendly biomaterials that are controlled by cell attachment timing. We selected poly-l-lysine (PLL) with Matrigel as the best combination of biomaterials for cell-defined culture. We used 2 µL PLL to determine by titration the optimum concentration required (0.04% stock, 0.005% final concentration). We also determined the optimum concentration of 10 µL of lentivirus particles for maximum reverse infection efficiency (1 × 108 infectious units [IFU]/mL stock, 62.5% final concentration) and established the best combination of components for the lentivirus mixture (10 µL of lentivirus particles and 2 µL each of siGLO Red dye, Matrigel, and 0.04% PLL). Finally, we validated both the effect of reverse infection in various cell lines and lentivirus spot activity in CDLM by storage period. This method provides an effective lentivirus-infected cell microarray for large-scale gene function studies.
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Lentivirus/metabolismo , Mamíferos/metabolismo , Análisis por Micromatrices/métodos , Animales , Materiales Biocompatibles/farmacología , Línea Celular Tumoral , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Polilisina/química , Reproducibilidad de los ResultadosRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is the most challenging type of cancer to treat, with a 5-year survival rate of <10%. Furthermore, because of the large portion of the inoperable cases, it is difficult to obtain specimens to study the biology of the tumors. Therefore, a patient-derived xenograft (PDX) model is an attractive option for preserving and expanding these tumors for translational research. Here we report the generation and characterization of 20 PDX models of PDAC. The success rate of the initial graft was 74% and most tumors were re-transplantable. Histological analysis of the PDXs and primary tumors revealed a conserved expression pattern of p53 and SMAD4; an exome single nucleotide polymorphism (SNP) array and Comprehensive Cancer Panel showed that PDXs retained over 94% of cancer-associated variants. In addition, Polyphen2 and the Sorting Intolerant from Tolerant (SIFT) prediction identified 623 variants among the functional SNPs, highlighting the heterologous nature of pancreatic PDXs; an analysis of 409 tumor suppressor genes and oncogenes in Comprehensive Cancer Panel revealed heterologous cancer gene mutation profiles for each PDX-primary tumor pair. Altogether, we expect these PDX models are a promising platform for screening novel therapeutic agents and diagnostic markers for the detection and eradication of PDAC.
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Trasplante de Neoplasias , Neoplasias Pancreáticas/genética , Polimorfismo de Nucleótido Simple , Animales , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Exoma , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Variación Genética , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Análisis Multivariante , Mutación , Neoplasias Pancreáticas/patología , República de Corea , Transducción de Señal , Proteína Smad4/genética , Proteína p53 Supresora de Tumor/genética , Neoplasias PancreáticasRESUMEN
Liver cancer is one of the common malignancies in many countries and an increasing cause of cancer death. Despite of that, there are few therapeutic options available with inconsistent outcome, raising a need for developing alternative therapeutic options. Through a drug repositioning screening, we identified and investigated the action mechanism of the Riluzole, an amyotrophic lateral sclerosis (ALS) drug, on hepatocellular carcinoma (HCC) therapy. Treatment of the Riluzole leads to a suppression of cell proliferation in liver primary cancer cells and cancer cell lines. In addition, Riluzole induced caspase-dependent apoptosis and G2/M cell cycle arrest in SNU449 and Huh7 cell lines. In a line with the known function of glutamate release inhibitor, we found Riluzole-treated cells have increased the level of inner cellular glutamate that in turn decrease the glutathione (GSH) level and finally augment the reactive oxygen species (ROS) production. We confirm this finding in vivo by showing the Riluzole-induced GSH and ROS changes in a Huh7 xenograft cancer model. Altogether, these data suggest the anti-cancer effect of Riluzole on hepatocellular carcinoma and the suppression of glutamate signaling might be a new target pathway for HCC therapy.
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Antineoplásicos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Proliferación Celular/efectos de los fármacos , Antagonistas de Aminoácidos Excitadores/farmacología , Ácido Glutámico/metabolismo , Neoplasias Hepáticas/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Riluzol/farmacología , Animales , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Reposicionamiento de Medicamentos , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Glutatión/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Células Tumorales Cultivadas , Regulación hacia Arriba , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: To effectively use pancreatic cancer patient-derived xenograft (PDX) models in translational research, successful PDX engraftment of surgical specimens in immune-deficient mice is needed. MATERIALS AND METHODS: A total of 102 patients underwent pancreatic cancer resection using various procedures. Tumor tissue from all patents was implanted subcutaneously into mice. Tumor engraftment and growth in mice were determined. Engraftment was tested for correlation with operation type, time, tumor size, and oncogene expression using immunohistoculture. RESULTS: Multivariate analysis showed that a tumor size of more than 3.5 cm in the patient was a significant factor related to successful PDX engraftment. In contrast, there was no correlation of engraftment with surgical procedure, time needed to remove the specimen, tumor differentiation, lymph node metastasis, and protein expression of p53, Receptor tyrosine-protein kinase erbB-2 (CERBB2), or deleted in pancreatic carcinoma locus 4 (DPC4). CONCLUSION: A minimum tumor size in the patient is an important factor for successful tumor engraftment.
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Adenocarcinoma/patología , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/patología , Adenocarcinoma/metabolismo , Adenocarcinoma/cirugía , Animales , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/cirugía , Femenino , Estudios de Seguimiento , Humanos , Técnicas para Inmunoenzimas , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Estadificación de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/cirugía , Pronóstico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor-initiating cells are important for the formation and maintenance of tumor bulks in various tumors. To identify surface markers of liver tumor-initiating cells, we performed primary tumorsphere culture and analyzed the expression of cluster of differentiation (CD) antigen genes using NanoString. Interestingly, we found significant upregulation of the complement proteins (p = 1.60 × 10(-18)), including C7 and CFH. Further studies revealed that C7 and CFH are required to maintain stemness in liver cancer cells. Knockdown of C7 and CFH expression abrogated tumorsphere formation and induced differentiation, whereas overexpression stimulated stemness factor expression as well as in vivo cell growth. Mechanistically, by studying C7 and CFH-dependent LSF-1 expression and its direct role on stemness factor transcription, we found that LSF-1 is involved in this regulation. Taken together, our data demonstrate the unprecedented role of complement proteins on the maintenance of stemness in liver tumor-initiating cells.
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Diferenciación Celular , Proliferación Celular , Complemento C7/metabolismo , Proteínas de Unión al ADN/metabolismo , Neoplasias Hepáticas/metabolismo , Células Madre Neoplásicas/metabolismo , Factores de Transcripción/metabolismo , Adulto , Anciano , Animales , Línea Celular Tumoral , Complemento C7/genética , Complemento C7/inmunología , Factor H de Complemento/genética , Factor H de Complemento/inmunología , Factor H de Complemento/metabolismo , Proteínas de Unión al ADN/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Xenoinjertos , Humanos , Inmunidad Innata , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/patología , Masculino , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Células Madre Neoplásicas/inmunología , Células Madre Neoplásicas/patología , Fenotipo , Interferencia de ARN , Transducción de Señal , Esferoides Celulares , Factores de Tiempo , Factores de Transcripción/genética , Transfección , Carga Tumoral , Células Tumorales CultivadasRESUMEN
The role of microRNAs (miRNAs) in carcinogenesis as tumor suppressors or oncogenes has been widely reported. Epigenetic change is one of the mechanisms of transcriptional silencing of miRNAs in cancer. To identify lung cancer-related miRNAs that are mediated by histone modification, we conducted microarray analysis in the Calu-6 non-small cell lung cancer (NSCLC) cell line after treatment with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase (HDAC) inhibitor. The expression level of miR-373 was enhanced by SAHA treatment in this cell line by microarray and the following quantitative RT-PCR analyses. Treatment with another HDAC inhibitor, Trichostatin A, restored the levels of miR-373 expression in A549 and Calu-6 cells, while demethylation drug treatment did not. Importantly, miR-373 was found to be down-regulated in NSCLC tissues and cell lines. Transfection of miR-373 into A549 and Calu-6 cells attenuated cell proliferation, migration, and invasion and reduced the expression of mesenchymal markers. Additional microarray analysis of miR-373-transfected cells and computational predictions identified IRAK2 and LAMP1 as targets of miR-373. Knockdown of these two genes showed similar biological effects to those of miR-373 overexpression. In clinical samples, overexpression of IRAK2 correlated with decreased disease-free survival of patients with non-adenocarcinoma. In conclusion, we found that miR-373 is silenced by histone modification in lung cancer cells and identified its function as a tumor suppressor and negative regulator of the mesenchymal phenotype through downstream IRAK2 and LAMP1 target genes.
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Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Epigénesis Genética , Quinasas Asociadas a Receptores de Interleucina-1/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Membrana de los Lisosomas/metabolismo , MicroARNs/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/mortalidad , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Supervivencia sin Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Regulación Neoplásica de la Expresión Génica , Histonas/metabolismo , Humanos , Estimación de Kaplan-Meier , Neoplasias Pulmonares/mortalidad , Neoplasias Pulmonares/patología , Masculino , Análisis Multivariante , Modelos de Riesgos Proporcionales , Procesamiento Proteico-PostraduccionalRESUMEN
Cultures of primary tumors are very useful as a personalized screening system for effective therapeutic options. We here describe an effective method of reproducing human primary colon tumors through primary culture and a mouse xenograft model. A total of 199 primary colon tumor cultures were successfully established under optimized conditions to enrich for tumor cells and to expand it for long-term storage in liquid nitrogen. To examine whether these stored cultures retained original tumor properties, fifty primary cultures were xenografted into NOD-SCID mouse. Histological and tumor marker analysis of four representative tumor xenografts revealed that all of the xenograft retained its primary tumor characteristics. Oncomap analysis further showed no change in the major mutations in the xenografts, confirming that our method faithfully reproduced human colon tumors. A drug sensitivity assay revealed that two of the primary cultures were hypersensitive to oxaliplatin rather than 5-FU, which was used in the patients, suggesting it as an effective therapeutic option. We thus present an effective, reproducible preclinical model for testing various personalized therapeutic options in colon cancer patients.
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Neoplasias del Colon/tratamiento farmacológico , Neoplasias del Colon/genética , Mutación , Ensayos Antitumor por Modelo de Xenoinjerto/métodos , Adulto , Anciano , Animales , Biomarcadores de Tumor/análisis , Neoplasias del Colon/patología , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos , Femenino , Genotipo , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Resultado del Tratamiento , Adulto JovenRESUMEN
We screened a chemical library in MCF-7 cells stably expressing green fluorescent protein (GFP)-conjugated microtubule-associated protein 1 light chain 3 (LC3) (GFP-LC3-MCF-7) using cell-based assay, and identified BIX-01294 (BIX), a selective inhibitor of euchromatic histone-lysine N-methyltransferase 2 (EHMT2), as a strong autophagy inducer. BIX enhanced formation of GFP-LC3 puncta, LC3-II, and free GFP, signifying autophagic activation. Inhibition of these phenomena with chloroquine and increasement in punctate dKeima ratio (550/438) signal indicated that BIX activated autophagic flux. BIX-induced cell death was suppressed by the autophagy inhibitor, 3-methyladenine, or siRNA against BECN1 (VPS30/ATG6), ATG5, and ATG7, but not by caspase inhibitors. Moreover, EHMT2 siRNA augmented GFP-LC3 puncta, LC3-II, free GFP, and cell death, implying that inhibition of EHMT2 caused autophagy-mediated cell death. Treatment with EHMT2 siRNA and BIX accumulated intracellular reactive oxygen species (ROS). BIX augmented mitochondrial superoxide via NADPH oxidase activation. In addition, BIX increased hydrogen peroxide and glutathione redox potential in both cytosol and mitochondria. Treatment with N-acetyl-L-cysteine (NAC) or diphenyleneiodonium chloride (DPI) decreased BIX-induced LC3-II, GFP-LC3 puncta, and cell death, indicating that ROS instigated autophagy-dependent cell death triggered by BIX. We observed that BIX potentiated autophagy-dependent and caspase-independent cell death in estrogen receptor (ESR)-negative SKBr3 and ESR-positive MCF-7 breast cancer cells, HCT116 colon cancer cells, and importantly, in primary human breast and colon cancer cells. Together, the results suggest that BIX induces autophagy-dependent cell death via EHMT2 dysfunction and intracellular ROS accumulation in breast and colon cancer cells, therefore EHMT2 inhibition can be an effective therapeutic strategy for cancer treatment.
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Autofagia/efectos de los fármacos , Azepinas/farmacología , Inhibidores Enzimáticos/farmacología , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Quinazolinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Células Cultivadas , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Femenino , Células HCT116 , Humanos , Células MCF-7 , Ratones , Cultivo Primario de Células , Transducción de Señal/efectos de los fármacosRESUMEN
Identification of gastric tumor-initiating cells (TICs) is essential to explore new therapies for gastric cancer patients. There are reports that gastric TICs can be identified using the cell surface marker CD44 and that they form floating spheres in culture, but we could not obtain consistent results with our patient-derived tumor xenograft (PDTX) cells. We thus searched for another marker for gastric TICs, and found that CD49f(high) cells from newly-dissected gastric cancers formed tumors with histological features of parental ones while CD49f(low) cells did not when subcutaneously injected into immunodeficient mice. These results indicate that CD49f, a subunit of laminin receptors, is a promising marker for human gastric TICs. We established a primary culture system for PDTX cells where only CD49f(high) cells could grow on extracellular matrix (ECM) to form ECM-attaching spheres. When injected into immunodeficient mice, these CD49f(high) sphere cells formed tumors with histological features of parental ones, indicating that only TICs could grow in the culture system. Using this system, we found that some sphere-forming TICs were more resistant than gastric tumor cell lines to chemotherapeutic agents, including doxorubicin, 5-fluorouracil and doxifluridine. There was a patient-dependent difference in the tumorigenicity of sphere-forming TICs and their response to anti-tumor drugs. These results suggest that ECM plays an essential role for the growth of TICs, and that this culture system will be useful to find new drugs targeting gastric TICs.
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Biomarcadores de Tumor/metabolismo , Integrina alfa6/metabolismo , Células Madre Neoplásicas/metabolismo , Esferoides Celulares/metabolismo , Neoplasias Gástricas/metabolismo , Animales , Antineoplásicos/farmacología , Doxorrubicina/farmacología , Resistencia a Antineoplásicos , Floxuridina/farmacología , Fluorouracilo/farmacología , Humanos , Concentración 50 Inhibidora , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Trasplante de Neoplasias , Neoplasias Gástricas/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: Preclinical trials of cancer therapeutics require both in vitro and in vivo evaluations. Recently, a patient-derived xenograft model in immunodeficient mice has been reported as a valuable in vivo evaluation system. In our current study, we aimed to establish a more efficient and accurate system for preclinical trials by generating primary cancer cells from patients and performing xenograft transfers of these cells into mice. METHODS: Human lung cancer specimens (n = 4) obtained from chemo-naive patients were cultured in bronchiolar epithelial basal medium supplemented with growth factors, followed by inoculation into non-obese diabetic/severe combined immunodeficient mice. The generated tumors in the mice were validated phenotypically and genetically using the original specimen and primary cancer cells. RESULTS: Immunohistochemical analysis of marker proteins, including cytokeratin 7, cytokeratin 20, epidermal growth factor receptor, thyroid transcription factor-1, CD56, chromogranin, and synaptophysin, demonstrated that the xenograft tumors were originated from the patient tumors. Moreover, mutation profiling using the OncoMap System, which analyzes mutations at 440 sites in 41 tumor-related genes, showed the same patterns in both the patient and xenograft tumors. CONCLUSIONS: These results indicate that our animal system is suitable for the amplification of patient tumors and will therefore be beneficial for both in vivo and in vitro assessments and preclinical trials of chemotherapeutics. This has the potential to provide a very effective tool for future personalized therapy and for conducting translational lung cancer research.
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Adenocarcinoma/patología , Biomarcadores de Tumor/análisis , Carcinoma Adenoescamoso/patología , Modelos Animales de Enfermedad , Neoplasias Pulmonares/patología , Carcinoma Pulmonar de Células Pequeñas/patología , Ensayos Antitumor por Modelo de Xenoinjerto , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Adulto , Anciano , Animales , Carcinoma Adenoescamoso/genética , Carcinoma Adenoescamoso/metabolismo , Femenino , Humanos , Inmunofenotipificación , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Persona de Mediana Edad , Mutación/genética , Fenotipo , Cultivo Primario de Células , Carcinoma Pulmonar de Células Pequeñas/genética , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Células Tumorales CultivadasRESUMEN
The umbilical cord contains mucinous connective tissue, called Wharton's jelly. It consists of stromal cells, collagen fibers, and amorphous ground substances composed of proteoglycan. Recently, these stromal cells have been redefined as a new cell therapy source, named human umbilical cord-derived mesenchymal stromal cells (hUCMSCs). However, there are few studies on the ultrastructural features and immune-phenotypic characteristics of isolated hUCMSCs and comparisons with the cells found in original cord tissues. In this study, the authors describe and compare the phenotypic characteristics of hUCMSCs with cells in the umbilical cord in order to know the kinds of cells and ultrastructural changes. Isolated hUCMSCs showed similar ultrastructure with few structural differences from in situ stromal cells, and they are relatively homogenous and well-developed mesenchymal cells that demonstrate a myofibroblastic phenotype.
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Inmunofenotipificación , Células Madre Mesenquimatosas/inmunología , Células Madre Mesenquimatosas/ultraestructura , Cordón Umbilical/inmunología , Cordón Umbilical/ultraestructura , Gelatina de Wharton/inmunología , Gelatina de Wharton/ultraestructura , Biomarcadores/análisis , Separación Celular/métodos , Células Cultivadas , Femenino , Humanos , Inmunohistoquímica , Microscopía Electrónica de Transmisión , Miofibroblastos/inmunología , Miofibroblastos/ultraestructura , Fenotipo , Embarazo , Cordón Umbilical/citología , Gelatina de Wharton/citologíaRESUMEN
BACKGROUND: Microarray analyses have revealed significantly elevated expression of the proto-oncogene ROS1 receptor tyrosine kinase in 20-30% of non-small cell lung carcinomas (NSCLC). Selective and potent ROS1 kinase inhibitors have recently been developed and oncogenic rearrangement of ROS1 in NSCLC identified. METHODS: We performed immunohistochemical evaluation of expression of ROS1 kinase and its downstream molecules in 399 NSCLC cases. ROS1 expression in primary and recurring lesions of 92 recurrent NSCLC cases was additionally analyzed. To elucidate mechanism of expression, two ROS1-nonexpressing NSCLC cell lines (Calu6 and H358) and fresh frozen tissues from 28 consecutive NSCLC patients were examined for ROS1 promoter methylation status and ROS1 expression. RESULTS: Overall expression rate of ROS1 was 22% (19% for adenocarcinomas and 25% for nonadenocarcinomas) in NSCLC. ROS1 expression was a worse prognostic factor for overall survival in adenocarcinomas of stage I NSCLC. In recurred NSCLC, ROS1 expression was significantly higher in recurring tumors (38%) than primary tumors (19%). Two NSCLC cell lines showed increased ROS1 expression after treatment with 5-aza-2'deoxycytidine and/or trichostatin A. Among the 14 adenocarcinomas examined, two (14%) showed more than twice the level of ROS1 expression in tumor tissue than was observed in matched normal tissue and statistically significant differences in the ROS1 promoter methylation level. CONCLUSIONS: A subset of NSCLC revealed overexpression of ROS1 receptor tyrosine kinase, possibly in relation to epigenetic changes. ROS1 expression was an independent prognostic factor for overall survival in adenocarcinomas of stage I NSCLC. Further studies are needed to validate our results.
Asunto(s)
Adenocarcinoma/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Neoplasias Pulmonares/metabolismo , Recurrencia Local de Neoplasia/metabolismo , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Adenocarcinoma/patología , Carcinoma de Pulmón de Células no Pequeñas/patología , Línea Celular Tumoral , Distribución de Chi-Cuadrado , Metilación de ADN , Epigénesis Genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Estimación de Kaplan-Meier , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/genética , Regiones Promotoras Genéticas , Modelos de Riesgos Proporcionales , Proto-Oncogenes Mas , Proteínas Proto-Oncogénicas c-vav/metabolismo , Factor de Transcripción STAT3/metabolismoRESUMEN
Wild-type p53-induced phosphatase 1 (Wip1, PPM1D) is induced by p53 in response to various stressors and dephosphorylates cellular target proteins involved in DNA repair and cell cycle checkpoint pathways. The Wip1 gene is frequently amplified or overexpressed in human cancers, promoting tumor growth by switching off major checkpoint kinases and p53. To explore wild-type p53-independent Wip1 induction, Wip1 promoter activity and its transcript level were evaluated by luciferase assay and real-time PCR, after methylmethane sulfonate (MMS) treatment in breast cancer cell lines and p53-null cell lines. Wip1 promoter activities in response to UV irradiation and various anti-cancer agents were compared between wild-type and a p53-response element (p53RE) mutated construct. Wip1 expression and its effects were examined in primary non-small cell lung cancer (NSCLC) and colon tumor cells by using Wip1-specific siRNA. MMS induced Wip1 promoter activity in Hs578T, MDA-MB-231, and SK-BR-3 cells expressing DNA binding-deficient p53 mutants. A549-E6 and HCT116 (p53(-/-)) cells retained substantial Wip1 induction. Wip1 promoter activity was reduced, but not eliminated, in cells expressing a promoter containing a mutated p53-response element. Wip1 induction was not blocked by SB202190 or SP600125. MMS increased Wip1 expression in primary non-small cell lung cancer cells expressing a p53 R175H mutant. Our data indicate that Wip1 is induced in the absence of functional p53, like p38 MAPK and JNK, as a stress response terminator.
Asunto(s)
Metilmetanosulfonato/farmacología , Neoplasias/tratamiento farmacológico , Fosfoproteínas Fosfatasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Secuencia de Bases , Western Blotting , Línea Celular Tumoral , Cartilla de ADN , Silenciador del Gen , Humanos , Neoplasias/enzimología , Neoplasias/metabolismo , Fosfoproteínas Fosfatasas/genética , Regiones Promotoras Genéticas , Proteína Fosfatasa 2C , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The interferon-inducible Sp100 proteins are thought to play roles in the chromatin pathway and in transcriptional regulation. Sp100A, the smallest isoform, is one of the major components of PML nuclear bodies (NBs) that exhibit intrinsic antiviral activity against several viruses. Since PML NBs are disrupted by the immediate-early 1 (IE1) protein during human cytomegalovirus (HCMV) infection, the modulation of Sp100 protein expression or activity during infection has been suggested. Here, we show that Sp100 proteins are lost largely in the late stages of HCMV infection. This event required viral gene expression and involved posttranscriptional control. The mutant virus with deletion of the sequence for IE1 (CR208) did not have Sp100 loss. In CR208 infection, PML depletion by RNA interference abrogated the accumulation of SUMO-modified Sp100A and of certain high-molecular-weight Sp100 isoforms but did not significantly affect unmodified Sp100A, suggesting that the IE1-induced disruption of PML NBs is not sufficient for the complete loss of Sp100 proteins. Sp100A loss was found to require proteasome activity. Depletion of all Sp100 proteins by RNA silencing enhanced HCMV replication and major IE (MIE) gene expression. Sp100 knockdown enhanced the acetylation level of histones associated with the MIE promoter, demonstrating that the repressive effect of Sp100 proteins may involve, at least in part, the epigenetic control of the MIE promoter. Sp100A was found to interact directly with IE1 through the N-terminal dimerization domain. These findings indicate that the IE1-dependent loss of Sp100 proteins during HCMV infection may represent an important requirement for efficient viral growth.
Asunto(s)
Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Citomegalovirus/patogenicidad , Expresión Génica , Interacciones Huésped-Patógeno , Proteínas Inmediatas-Precoces/metabolismo , Replicación Viral , Células Cultivadas , Citomegalovirus/crecimiento & desarrollo , Silenciador del Gen , HumanosRESUMEN
The triglyceride lipase gene family, including lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL), carboxyl ester lipase (CEL), endothelial lipase (EL), Lipase H, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), plays a critical role in lipid metabolism in mammals. In this study, we have identified and characterized the expression profile of these genes in the chicken, Gallus gallus. Chicken LPL and ATGL have been cloned, and HTGL, EL, Lipase H, and CEL sequences were found in the chicken genome database. The deduced amino acid sequences of HTGL, EL, Lipase H, and CEL were 66, 75, 63, and 65% identical with their respective human genes, suggesting conservation of important enzymatic functions. In contrast, a homologue of the HSL gene was not identified in the chicken genome. We performed RT-PCR using chicken liver, muscle, abdominal adipose tissue, or pancreas mRNA as the template, and all partial products were completely matched to the corresponding predicted sequences of triglyceride lipase gene members. Quantitation by qPCR of the transcript levels of these genes in 13 tissues indicates that the expression patterns diverge greatly between species. A particularly interesting pattern was observed in the distribution of EL and HTGL mRNA, which were highly expressed in kidney and ovary. This is the first report of HTGL, EL, Lipase H, and CEL in a pre-mammalian species and reveals novel details about specific features of the expression of these important molecules in lipid metabolism.
Asunto(s)
Pollos/genética , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Lipasa/genética , Grasa Abdominal/enzimología , Grasa Abdominal/metabolismo , Animales , Femenino , Humanos , Riñón/enzimología , Riñón/metabolismo , Lipoproteína Lipasa/genética , Hígado/enzimología , Hígado/metabolismo , Músculo Esquelético/enzimología , Músculo Esquelético/metabolismo , Ovario/enzimología , Ovario/metabolismo , Páncreas/enzimología , Páncreas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esterol Esterasa/genéticaRESUMEN
Angiopoietin-like protein 3 (ANGPTL3) is a 70 kDa secretory glycoprotein, which has been identified as a plasma triacylglycerol (TG)-increasing factor in KK/San mice. In this study, we have characterized the expression profile and activity of ANGPTL3 in the chicken, Gallus gallus. This is the first report of this protein in a pre-mammalian species and reveals novel details about the molecular structure of, and specific features of the expression of, important molecules in lipid metabolism. Recombinant chicken ANGPTL3 inhibited chicken lipoprotein lipase activity in vitro and increased plasma TG concentrations in vivo. Chicken ANGPTL3 mRNA expression was detected only in the liver. ANGPTL3 mRNA expression was significantly higher in mature (i.e. egg producing) chickens than in immature chickens. However, this increase was not associated with estradiol, although estradiol does enhance triacylglycerol and very low density lipoprotein secretion by the chicken liver. Changes in chicken ANGPTL3 mRNA expression with nutritional state were examined. ANGPLT3 expression was affected by certain nutritional treatments, i.e. fasting and re-feeding, and ANGPTL3 gene expression in the livers of chickens fed a cholesterol-supplemented diet was lower than that in the livers of chickens fed a control diet. The present study is the first to molecularly characterize and analyze the gene expression of ANGPTL3, and suggests that in avian species ANGPTL3 plays a key role in lipid delivery to the yolk.