RESUMEN
Coiled-coil domain containing 110 (CCDC110, KM-HN-1) is a protein containing C-terminal coiled-coil domain (CCD) which was previously discovered as a member of the human cancer/testis antigen (CTA). In addition, CCDC110 has both nuclear localization signal sequence and the leucine zipper motif. Although the functional role of CCDC110 has yet to be fully identified, the mRNA expression levels of CCDC110 are known to be highly elevated in various cancer types including testis, implying its relevance to cancer pathogenesis. In this study, we first developed several monoclonal antibody (mAb) hybridoma clones targeting CCDC110 and further isolated clone by characterizing for its specificity using immunoblotting and immunoprecipitation approaches with basal parenchymal sperm cells in testis tissue. Next, using these mAbs, we showed that the Tet-inducible overexpression of CCDC110 protein delayed the entry of G2/M phase in U2-OS osteosarcoma cells. Based on these results, we propose that CCDC110 plays a crucial role in cell cycle progression.
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Triple-negative breast cancer (TNBC) cells frequently exhibit activated growth factor signaling and resistance to inhibitors for epidermal growth factor receptor (EGFR), despite the overexpression of EGFR protein, and this is associated with a malignant behavior and a poor prognosis. In this study, to elucidate the underlying mechanisms of resistance to EGFR inhibitor and identify inhibitors that exert a synergistic effect with EGFR inhibition, we examined the inhibitory effects of selected protein kinase inhibitors (PKIs) in combination with gefitinib on the viability of a mesenchymal stem-like (MSL) subtype TNBC cell line. MK2206, an AKT inhibitor, and a group of mammalian target of rapamycin (mTOR) inhibitors were found to exert synergistic lethal effects in combination with gefitinib in MDAMB231 cells. The combination of gefitinib/MK2206 exerted a prominent synergistic lethal effect in an MTT cell viability assay and a growth inhibitory effect in a long-term colony-forming assay in 2 MSL subtype TNBC cell lines (MDAMB231 and HS578T) and one basal-like (BL) subtype TNBC cell line (MDAMB-468). Gefitinib/MK2206 treatment synergistically decreased the mTOR signaling target substrates along with the downregulation of ribosomal protein S6 (RPS6), a marker of cell proliferation and target substrate of the AKT-mTOR signaling pathway. In addition, gefitinib markedly reduced the viability of MDAMD231 and HS578T cells when regulatory-associated protein of mTOR (RPTOR) was suppressed by siRNA-based knockdown (KD). These results thus suggest that RPTOR mediates, at least partially, the resistance to EGFR inhibition in TNBC cells. Therefore, targeting the mTOR complex 1 (mTORC1) pathway may be a potential strategy for the treatment of EGFR-resistant TNBC.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Proteína Reguladora Asociada a mTOR/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Ensayos de Selección de Medicamentos Antitumorales , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Gefitinib , Técnicas de Silenciamiento del Gen , Compuestos Heterocíclicos con 3 Anillos/farmacología , Humanos , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Quinazolinas/farmacología , Quinazolinas/uso terapéutico , ARN Interferente Pequeño/metabolismo , Proteína Reguladora Asociada a mTOR/genética , Proteína S6 Ribosómica/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Neoplasias de la Mama Triple Negativas/patologíaRESUMEN
Pancreatic cancer remains an intractable cancer with a poor five-year survival rate, which requires new therapeutic modalities based on the biology of pancreatic oncogenesis. Nuclear factor E2 related factor-2 (NRF2), a key cytoprotective nuclear transcription factor, regulates antioxidant production, reduction, detoxification and drug efflux proteins. It also plays an essential role in cell homeostasis, cell proliferation and resistance to chemotherapy. We aimed to evaluate the possibility that modulation of NRF2 expression could be effective in the treatment of pancreatic cancer cells. We investigated whether the depletion of NRF2 by using small interfering RNAs (siRNAs) is effective in the expression of biomarkers of pancreatic cancer stemness such as aldehyde dehydrogenase 1 family, member A1 (ALDH1A1) and aldehyde dehydrogenase 3 family, member A1 (ALDH3A1). NRF2 knockdown markedly reduced the expression of NRF2 and glutamate-cysteine ligase catalytic subunit (GCLC) in cell lines established from pancreatic cancers. NRF2 silencing also decreased the ALDH1A1 and ALDH3A1 expression. Furthermore, this NRF2 depletion enhanced the antiproliferative effects of the chemotherapeutic agent, 5-fluorouracil (5-FU) in pancreatic cancer cells.
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The poly(ADP-ribose) polymerase (PARP) inhibitor, olaparib has been reported as having preferential anti-proliferative effects on breast cancer 1 (BRCA1)-deficient breast and ovarian cancer cells and was recently approved by the US Food and Drug Administration (FDA) for advanced, BRCA1-mutated ovarian cancer. Herein, we show that BEZ235, a protein kinase inhibitor, enhanced the tumor cell-killing effect of olaparib in BRCA1-mutated breast cancer cells in vitro. BEZ235 reduced olaparib-induced phosphorylation of p53 binding protein 1 (53BP1) and 53BP1 foci formation, as well as phosphorylation of AKT (S473). Long-term colony-formation assay revealed more strong synergistic effects of this combination in SUM149PT and MDA-MB-468 breast cancer cell lines. BEZ235 treatment combined with olaparib may be a candidate for effective therapeutic treatment of BRCA1-mutated breast cancer.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Proteína BRCA1/metabolismo , Neoplasias de la Mama/tratamiento farmacológico , Antineoplásicos/administración & dosificación , Neoplasias de la Mama/metabolismo , Línea Celular Tumoral , Inhibidores Enzimáticos/administración & dosificación , Femenino , Humanos , Imidazoles/administración & dosificación , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosforilación/efectos de los fármacos , Ftalazinas/administración & dosificación , Piperazinas/administración & dosificación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Quinolinas/administración & dosificación , Receptor ErbB-2/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Triple-negative breast cancer (TNBC) exhibits innate resistance to the EGFR inhibition despite high level expression of EGFR. Recently, we found that the proliferation of basal-like (BL) subtype TNBC cells is synergistically inhibited by combination of EGFR and PI3K/AKT inhibitors. On the contrary, TNBC cells of mesenchymal stem-like (MSL) subtype are resistant to these combinations. To identify potential synthetic lethal interaction of compounds for treatment of MSL subtype TNBC cells, we performed MTT screening of MDA-MB231 cells with a small library of receptor tyrosine kinase inhibitors (RTKIs) in the presence of gefitinib, an EGFR inhibitor. We identified MET inhibitors as potent RTKIs that caused synthetic lethality in combination with gefitinib in MDA-MB231 cells. We demonstrated that combination of a MET inhibitor SU11274 with various EGFR inhibitors resulted in synergistic suppression of cell viability (in MTT assay) and cell survival (in colony formation assay) of MSL subtype TNBC cells. We further demonstrated that SU11274 alone induced G2 arrest and gefitinib/SU11274 combination sustained the SU11274-induced G2 arrest in these cells. In addition, SU11274/gefitinib combination synergistically reduced the level of ribosomal protein S6 (RPS6) in MSL subtype TNBC cells. In addition, knockdown of RPS6 itself, in both HS578T and MDA-MB231, markedly reduced the proliferation of these cells. Taken together, our data suggest that dual targeting of EGFR and MET inhibits the proliferation of MSL subtype TNBC cells through downregulation of RPS6.
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Receptores ErbB/antagonistas & inhibidores , Indoles/farmacología , Piperazinas/farmacología , Proteínas Proto-Oncogénicas c-met/antagonistas & inhibidores , Quinazolinas/farmacología , Proteína S6 Ribosómica/metabolismo , Sulfonamidas/farmacología , Neoplasias de la Mama Triple Negativas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación hacia Abajo , Sinergismo Farmacológico , Femenino , Gefitinib , Humanos , Biblioteca de Péptidos , Neoplasias de la Mama Triple Negativas/tratamiento farmacológicoRESUMEN
BACKGROUND: Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family. PRINCIPAL FINDINGS: NM II colocalized with GEFs, such as ßPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II-GEF interactions by overexpression of the DH domains of ßPIX or Tiam1, or by ßPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II-GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation. CONCLUSION: Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF-Rho GTPase signaling pathway.
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Actinas/metabolismo , Neuronas/metabolismo , Miosina Tipo IIB no Muscular/metabolismo , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Animales , Axones/efectos de los fármacos , Axones/metabolismo , Células Cultivadas , Femenino , Conos de Crecimiento/efectos de los fármacos , Conos de Crecimiento/metabolismo , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Hipocampo/citología , Modelos Biológicos , Factores de Crecimiento Nervioso/metabolismo , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuronas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Seudópodos/efectos de los fármacos , Seudópodos/metabolismo , Ratas Sprague-Dawley , Factores de Intercambio de Guanina Nucleótido Rho/química , Transducción de Señal/efectos de los fármacos , Proteína de Unión al GTP cdc42/metabolismo , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/metabolismoRESUMEN
Gemcitabine-based chemotherapy is the standard for treatment of pancreatic cancer; however, intrinsic and acquired resistance to gemcitabine commonly occurs. Aldehyde dehydrogenase 1A1 (ALDH1A1), one of the characteristic features of tumor-initiating and/or cancer stem cell (CSC) properties, is important in both intrinsic and acquired resistance to gemcitabine. In this study, we investigated the effectiveness of dasatinib, an SRC inhibitor, and gemcitabine combination to inhibit the survivals of parental (MIA PaCa-2/P) and gemcitabine-resistant (MIA PaCa-2/GR) cell lines. In MIA PaCa-2/GR cells, the levels of phospho-SRC and ALDH1A1 were increased compared to MIA PaCa-2/P cells. Inhibition of SRC by dasatinib or siRNA synergistically enhanced gemcitabine-induced anti-proliferative effects and induced apoptotic cell death in these cells. Furthermore, combination of SRC inhibition (either by dasatinib or siRNA) and gemcitabine significantly decreased the levels of ALDH1A1 expression. These results suggest that dasatinib and gemcitabine combination may be a potential therapeutic strategy to overcome gemcitabine resistance by decreasing the levels of ALDH1A1 expression.
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Aldehído Deshidrogenasa/metabolismo , Antineoplásicos/farmacología , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Pirimidinas/farmacología , Tiazoles/farmacología , Aldehído Deshidrogenasa/genética , Familia de Aldehído Deshidrogenasa 1 , Protocolos de Quimioterapia Combinada Antineoplásica , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Dasatinib , Desoxicitidina/farmacología , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Retinal-Deshidrogenasa , GemcitabinaRESUMEN
BACKGROUND: The ataxia-telangiectasia mutated (ATM) protein kinase plays a central role in coordinating the cellular response to radiation-induced DNA damage. cAMP signaling regulates various cellular responses including metabolism and gene expression. This study aimed to investigate the mechanism through which cAMP signaling regulates ATM activation and cellular responses to ionizing radiation in lung cancer cells. METHODS: Lung cancer cells were transfected with constitutively active stimulatory G protein (GαsQL), and irradiated with γ-rays. The phosphorylation of ATM and protein phosphatase 2A was analyzed by western blotting, and apoptosis was assessed by western blotting, flow cytometry, and TUNNEL staining. The promoter activity of NF-κB was determined by dual luciferase reporter assay. BALB/c mice were treated with forskolin to assess the effect in the lung tissue. RESULTS: Transient expression of GαsQL significantly inhibited radiation-induced ATM phosphorylation in H1299 human lung cancer cells. Treatment with okadaic acid or knock down of PP2A B56δ subunit abolished the inhibitory effect of Gαs on radiation-induced ATM phosphorylation. Expression of GαsQL increased phosphorylation of the B56δ and PP2A activity, and inhibition of PKA blocked Gαs-induced PP2A activation. GαsQL enhanced radiation-induced cleavage of caspase-3 and PARP and increased the number of early apoptotic cells. The radiation-induced apoptosis was increased by inhibition of NF-κB using PDTC or inhibition of ATM using KU55933 or siRNA against ATM. Pretreatment of BALB/c mice with forskolin stimulated phosphorylation of PP2A B56δ, inhibited the activation of ATM and NF-κB, and augmented radiation-induced apoptosis in the lung tissue. GαsQL expression decreased the nuclear levels of the p50 and p65 subunits and NF-κB-dependent activity after γ-ray irradiation in H1299 cells. Pretreatment with prostaglandin E2 or isoproterenol increased B56δ phosphorylation, decreased radiation-induced ATM phosphorylation and increased apoptosis. CONCLUSIONS: cAMP signaling inhibits radiation-induced ATM activation by PKA-dependent activation of PP2A, and this signaling mechanism augments radiation-induced apoptosis by reducing ATM-dependent activation of NF-κB in lung cancer cells.
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Proteínas de la Ataxia Telangiectasia Mutada/genética , AMP Cíclico/metabolismo , Neoplasias Pulmonares/genética , Proteína Fosfatasa 2/biosíntesis , Animales , Apoptosis/genética , Apoptosis/efectos de la radiación , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Línea Celular Tumoral , AMP Cíclico/genética , Daño del ADN/efectos de la radiación , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Ratones , Fosforilación/genética , Fosforilación/efectos de la radiación , Proteína Fosfatasa 2/metabolismo , Transducción de Señal/genética , Transducción de Señal/efectos de la radiaciónRESUMEN
The p90 ribosomal S6 kinase family (RSK1-4) of Ser/Thr kinases is a downstream component of the Ras-MAPK cascade responsible for regulating various cellular processes. Here, we examined the potential involvement of RSKs in regulating mitosis by transfecting HeLa cells with siRNAs targeting RSK1 and -2, which are the major isoforms. Depletion of RSK1 but not RSK2 triggered a significant accumulation of binucleated cells compared to control cells (0.5% vs. 10.5%, respectively); this was rescued by expression of exogenous RSK1 but not a kinase-defective mutant. Monitoring of cell division by time-lapse imaging revealed that the observed binucleation mainly stemmed from a failure to form and ingress the cleavage furrow during early cytokinesis. Immunocytochemical analysis of RhoA and anillin, the two principal regulators of cleavage furrow formation and ingression, showed that these proteins were abnormally localized during anaphase in RSK1-depleted cells. Furthermore, RSK1-depleted cells seemed to have impairments in midzone microtubule formation, as suggested by morphological changes and lengthening of the midzone (15.2 ± 1.7 µm vs. 17.4 ± 1.7 µm in control cells). We also observed shortening of the pole-to-polar-cortex distance in RSK1-depleted cells (4.30 ± 1.37 µm vs. 2.80 ± 0.84 µm in control cells) and scanty distribution of microtubules at the periphery of the equatorial region during anaphase, suggesting an aberrant distribution of astral microtubules. Taken together, these results suggest that RSK1 is specifically required for cleavage furrow formation and ingression during cytokinesis. This may occur via the involvement of RSK1 in proper midzone and astral microtubule structure formation during anaphase, which is essential for the correct localization of anillin and RhoA.
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Citocinesis/genética , Proteínas Quinasas S6 Ribosómicas 90-kDa/metabolismo , Anafase , Proteínas Contráctiles/metabolismo , Células HeLa , Humanos , Microtúbulos/metabolismo , Poliploidía , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteínas Quinasas S6 Ribosómicas 90-kDa/antagonistas & inhibidores , Proteínas Quinasas S6 Ribosómicas 90-kDa/genética , Imagen de Lapso de Tiempo , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
PURPOSE: The oil spill from the Heibei Spirit in December 2007 contaminated the Yellow Coast of South Korea. We evaluated the respiratory effects of that spill on children who lived along the Yellow Coast. METHODS: Of 662 children living in the area exposed to the oil spill, 436 (65.9%) were enrolled as subjects. All subjects completed a modified International Study of Asthma and Allergies in Childhood questionnaire. A health examination, including a skin prick test, pulmonary function test, and methacholine bronchial provocation test (MBPT), was administered. The children were assigned to two groups: those who lived close to the oil spill area and those who lived far from the oil spill area. RESULTS: The children who lived close to the oil spill area showed a significantly lower forced expiratory volume in one second (FEV1), an increased prevalence of 'asthma ever' (based on a questionnaire), and 'airway hyperresponsiveness' (based on the MBPT) than those who lived far from the oil spill area (FEV1; P=0.011, prevalence of 'asthma ever' based on a questionnaire; P=0.005, prevalence of 'airway hyperresponsiveness' based on the MBPT; P=0.001). The onset of wheezing after the oil spill was significantly higher in children who lived close to the oil spill area than in those who lived far from the oil spill area among the 'wheeze ever' group (P=0.002). In a multiple logistic regression analysis, male sex, family history of asthma, and residence near the oil spill area were significant risk factors for asthma (sex [male/female]: odds ratio [OR], 2.54; 95% confidence interval [CI], 1.31-4.91; family history of asthma [No/Yes]: OR, 3.77; 95% CI, 1.83-7.75; exposure group [low/high]; OR, 2.43; 95% CI, 1.27-4.65). CONCLUSIONS: This study suggests that exposure to an oil spill is a risk factor for asthma in children.
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During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS(-) mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS(+)p) complemented the phenotype of the GlmS(-) mutant, and that GlmS(+) p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS (+)pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number.
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Proteínas Bacterianas/genética , Escherichia coli/genética , Vectores Genéticos/genética , Neoplasias/genética , Neoplasias/microbiología , Plásmidos/genética , Salmonella typhimurium/genética , Animales , Línea Celular Tumoral , Células Cultivadas , Eliminación de Gen , Terapia Genética , Vectores Genéticos/administración & dosificación , Humanos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/terapia , Plásmidos/administración & dosificaciónRESUMEN
Gynaecotyla squatarolae (Digenea: Microphallidae) is a minute intestinal trematode whose natural hosts are aves. We conducted a feces screening survey in a coastal village of Muan-gun, where the residents routinely consume brackish water crabs as a food. Through this survey, a 50-year-old female was found to shed gymnophallid and heterophyid eggs in her stool, and 845 adult flukes were collected from her purged stool. The adult worms were morphologically grouped into three species. A total of 841 worms were Gymnophalloides seoi. Three worms were identified as G. squatarolae, and the last one proven to be Haplorchis pumilio. This is the first worldwide report of G. squatarolae infection in humans, and the first H. pumilio infection in Korean people.
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Heces/parasitología , Enfermedades Transmitidas por los Alimentos/parasitología , Heterophyidae/aislamiento & purificación , Infecciones por Trematodos/parasitología , Animales , Pueblo Asiatico , Braquiuros/parasitología , Femenino , Heterophyidae/clasificación , Humanos , Persona de Mediana Edad , Recuento de Huevos de Parásitos , República de CoreaRESUMEN
Microparticulates are an effective three-dimensional (3D) matrix for the culture of stem cells to be used in tissue engineering of bone. Herein, bioactive calcium phosphate microparticles with an evacuated morphology were prepared, and their potential to support stem cells for bone tissue engineering was addressed. Spherical particles with sizes of hundreds of micrometers were produced using the emulsification method, during which the internal portion was evacuated with the aid of solvent evaporation. The evacuated portion of the microspheres, which is considered to enhance cell population and be replaced with new bone, was found to comprise approximately 65-70% of the total volume. Stem cells derived from human adipose and rat bone marrow were isolated and cultured on the evacuated microspheres. When compared to a two-dimensional culture dish, the 3D spherical substrate provided cells with more space to adhere and populate, which became more evident as the cell seeding quantity increased. Moreover, better cell proliferation was observed on the evacuated microspheres than on the conventional filled microspheres, suggesting that evacuation of the internal part of the microspheres was useful for generating a large cell population. The differentiation of cells cultured on the 3D evacuated microspheres into osteoblasts with appropriate osteogenic cues was also more effective when compared to cells cultured on a two-dimensional dish. When implanted within a rabbit calvarium, the evacuated microspheres induced rapid bone formation at 6 weeks with a typical lamella pattern. Based on the results, the evacuated calcium phosphate microspheres are considered an effective 3D matrix for direct filling of bone defects as well as for bone tissue engineering using stem cells.
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Regeneración Ósea/efectos de los fármacos , Fosfatos de Calcio/farmacología , Portadores de Fármacos/química , Microesferas , Tejido Adiposo/citología , Animales , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Estudios de Factibilidad , Humanos , Osteogénesis/efectos de los fármacos , Proyectos Piloto , Conejos , Ratas , Cráneo/efectos de los fármacos , Cráneo/patología , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/ultraestructura , Ingeniería de Tejidos , Difracción de Rayos XRESUMEN
Angiotensin II (Ang II) stimulates migration of vascular smooth muscle cell (VSMC) in addition to its contribution to contraction and hypertrophy. It is well established that Rho GTPases regulate cellular contractility and migration by reorganizing the actin cytoskeleton. Ang II activates Rac1 GTPase, but its upstream guanine nucleotide exchange factor (GEF) remains elusive. Here, we show that Ang II-induced VSMC migration occurs in a betaPIX GEF-dependent manner. betaPIX-specific siRNA treatment significantly inhibited Ang II-induced VSMC migration. Ang II activated the catalytic activity of betaPIX towards Rac1 in dose- and time-dependent manners. Activity reached a peak at 10 min and declined close to a basal level by 30 min following stimulation. Pharmacological inhibition with specific kinase inhibitors revealed the participation of protein kinase C, Src family kinase, and phosphatidylinositol 3-kinase (PI3-K) upstream of betaPIX. Both p21-activated kinase and reactive oxygen species played key roles in cytoskeletal reorganization downstream of betaPIX-Rac1. Taken together, our results suggest that betaPIX is involved in Ang II-induced VSMC migration.
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Angiotensina II/metabolismo , Movimiento Celular , Factores de Intercambio de Guanina Nucleótido/metabolismo , Miocitos del Músculo Liso/citología , Animales , Células Cultivadas , Factores de Intercambio de Guanina Nucleótido/genética , Músculo Liso Vascular/citología , NADPH Oxidasas/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Factores de Intercambio de Guanina Nucleótido Rho , Quinasas p21 Activadas/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Familia-src Quinasas/metabolismoRESUMEN
Increased expression of a number of proinflammatory genes, including IL-8, is associated with inflammatory conditions such as asthma. Glucocorticoid receptor (GR)beta, one of the GR isoforms, has been suggested to be upregulated in asthma associated with glucocorticoid insensitivity and to work as a dominant negative inhibitor of wild type GRalpha. However, recent data suggest that GRbeta is not a dominant negative inhibitor of GRalpha in the transrepressive process and has its own functional role. We investigated the functional role of GRbeta expression in the suppressive effect of glucocorticoids on tumor necrosis factor (TNF)-alpha-induced IL-8 release in an airway epithelial cell line. GRbeta expression was induced by treatment of epithelial cells with either dexamethasone or TNF-alpha. GRbeta was able to inhibit glucocorticoid-induced transcriptional activation mediated by binding to glucocorticoid response elements (GREs). The suppressive effect of dexamethasone on TNF-alpha-induced IL-8 transcription was not affected by GRbeta overexpression, rather GRbeta had its own weak suppressive activity on TNF-alpha-induced IL-8 expression. Overall histone deacetylase activity and histone acetyltransferase activity were not changed by GRbeta overexpression, but TNF-alpha-induced histone H4 acetylation at the IL-8 promoter was decreased with GRbeta overexpression. This study suggests that GRbeta overexpression does not affect glucocorticoid-induced suppression of IL-8 expression in airway epithelial cells and GRbeta induces its own histone deacetylase activity around IL-8 promoter site.
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Regulación de la Expresión Génica , Histonas/metabolismo , Interleucina-8/genética , Receptores de Glucocorticoides/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Acetilación , Línea Celular Tumoral , Dexametasona/farmacología , Células Epiteliales/metabolismo , Humanos , Interleucina-8/metabolismo , Receptores de Glucocorticoides/genética , Activación Transcripcional , Transfección , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Mammalian polo-like kinase 1 (Plk1) has been studied intensively as a key element in regulating diverse mitotic events during M-phase progression. Plk1 is spatially regulated through the targeting activity of the conserved polo-box domain (PBD) present in the C-terminal non-catalytic region. Over the years, studies have demonstrated that the PBD forms a phospho-epitope binding module and the PBD-dependent interaction is critical for proper subcellular localization of Plk1. The current prevailing model is that the PBD binds to a phospho-epitope generated by Cdc2 or other Pro-directed kinases. Here we discuss a recent finding that Plk1 also self-promotes its localization by generating its own PBD-docking site.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Animales , Proteínas de Ciclo Celular/química , Humanos , Mitosis , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/química , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Huso Acromático/química , Quinasa Tipo Polo 1RESUMEN
BACKGROUND: Adipose-derived stem cells (ADSCs) are a population of pluripotent cells, which have characteristics similar to bone marrow-derived mesenchymal stem cells. Whereas ADSCs have potential applications for the repair and regeneration of various damaged tissues, few studies have dealt with the effect of ADSCs on fibroblasts, which play a key role in skin biology. OBJECTIVE: In this study, we investigated the possible roles of ADSCs in skin wound healing process, especially in the aspect of fibroblast activation-proliferation, collagen synthesis and migratory properties. METHODS AND RESULTS: ADSCs promoted human dermal fibroblast (HDF) proliferation, not only by cell-to-cell direct contact, which was confirmed by co-culture experiment, but also by paracrine activation through secretory factors, resolved by transwell co-culture and culturing with conditioned medium of ADSCs (ADSC-CM). ADSC-CM enhanced the secretion of type I collagen in HDFs by regulating the mRNA levels of extracellular matrix (ECM) proteins: up-regulation of collagen type I, III and fibronectin and down-regulation of MMP-1. Moreover, ADSC-CM showed stimulatory effect on migration of HDFs in in vitro wound healing models. Additional to those in vitro evidences, wound healing effect of ADSCs was also verified with in vivo animal study, resulted that ADSCs significantly reduced the wound size and accelerated the re-epithelialization from the edge. CONCLUSION: Collectively, these data suggest that ADSC is constitutionally well suited for dermal wound healing and secretory factors derived from ADSCs promote wound healing via HDFs and ADSCs can be used for the treatment of photoaging and wound healing.
Asunto(s)
Tejido Adiposo/citología , Dermis/citología , Fibroblastos/fisiología , Células Madre/fisiología , Cicatrización de Heridas/fisiología , Tejido Adiposo/metabolismo , Movimiento Celular/fisiología , Proliferación Celular , Células Cultivadas , Colágeno Tipo I/metabolismo , Colágeno Tipo III/metabolismo , Dermis/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/metabolismo , Femenino , Fibronectinas/metabolismo , Humanos , Metaloproteinasa 1 de la Matriz/metabolismo , Modelos Animales , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Cyclooxygenase-2 (COX-2) has been reported to be associated with tumor development and progression as well as to protect cells from apoptosis induced by various cellular stresses. Through a tetracycline-regulated COX-2 overexpression system, we found that COX-2 inhibits detachment-induced apoptosis (anoikis) in a human bladder cancer cell line, EJ. We also found that the inhibition of anoikis by COX-2 results from activation of the PI-3K/Akt pathway as evidenced by suppression of the COX-2 effect on anoikis by a PI-3K inhibitor, LY294002. Furthermore, COX-2 enhanced Mcl-1 expression in the anoikis process, implying that Mcl-1 also may be involved in mediating the survival function of COX-2. Together, these results suggest that COX-2 inhibits anoikis by activation of the PI-3K/Akt pathway and probably by enhancement of Mcl-1 expression in human bladder cancer cells. This anti- anoikis effect of COX-2 may be a part of mechanisms to promote tumor development and progression.
Asunto(s)
Anoicis/fisiología , Proteínas de Neoplasias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Neoplasias de la Vejiga Urinaria/metabolismo , Ciclooxigenasa 2 , Activación Enzimática , Humanos , Proteínas de la Membrana , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal , Transfección , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Mesenchymal stem cell (MSC) has drawn much attention in the aspect of tissue renewal and wound healing because of its multipotency. We initially observed that bone marrow-derived human MSCs (hMSCs) divided poorly and took flat and enlarged morphology after expanded in culture over a certain number of cell passage, which resembled characteristic features of senescent cells, well-studied in human diploid fibroblasts (HDFs). More interestingly, adipogenic differentiation potential of hMSCs sharply declined as they approached the end of their proliferative life span. In this study, altered hMSCs were verified to be senescent by their senescence-associated beta-galactosidase (SA-beta-gal) activity and the increased expression of cell cycle regulating proteins (p16(INK4a), p21(Waf1) and p53). Similar as in HDFs, basal phosphorylation level of ERK was also significantly increased in senescent hMSCs, implying altered signal paths commonly shared by the senescent cells. Insulin, a major component of adipogenesis inducing medium, did not phosphorylate ERK 1/2 more in senescent hMSCs after its addition whereas it did in young cells. In senescent hMSCs, we also found a significant increase of caveolin-1 expression, previously reported as a cause for the attenuated response to growth factors in senescent HDFs. When we overexpressed caveolin-1 in young hMSC, not only insulin signaling but also adipogenic differentiation was significantly suppressed with down-regulated PPARgamma2. These data indicate that loss of adipogenic differentiation potential in senescent hMSC is mediated by the over-expression of caveolin-1.
Asunto(s)
Adipocitos/citología , Caveolinas/metabolismo , Senescencia Celular/fisiología , Mesodermo/citología , Células Madre/citología , Células Madre/fisiología , Caveolina 1 , Diferenciación Celular/fisiología , Células Cultivadas , Humanos , Insulina/metabolismo , Transducción de Señal , Células Madre/metabolismoRESUMEN
Individual cell types express a characteristic balance between heterotrimeric G protein alpha and betagamma subunits, but little is known about the regulatory mechanism. We systemically examined the regulatory mechanism in CHO cells. We found that expression of Galphas, Galphai2, and Galphaq proteins increased in direct proportion to the increase of Gbeta1gamma2 overexpressed transiently. Expression of Gbeta protein also increased following overexpression of Galphas, Galphai2, and Galphaq. The Gbetagamma overexpression stimulated degradation of Gbeta in contrast to reduction of Galphas degradation. We conclude that coordinate expression of the G protein subunits involves regulation of protein degradation via proteasome in CHO cells.