RESUMEN
After review of the literature, there appears to be no report on the histology of the mandibular nerve fiber distribution. Therefore, using a Wistar rat model, immunohistochemical staining with protein gene product (PGP) and calcitonin gene-related peptide (CGRP) antibody for all nerves and only the pain-sensitive nerves, respectively, was performed. We also statistically compared the nerve distribution density by mandibular region. The section of the mandible from the alveolar crest to the mandibular canal was compartmentalized to several regions. Subsequently, nerve distribution density by region was measured microscopically in both the PGP- and CGRP-positive nerves. Furthermore, the ratio of CGRP- to PGP-positive nerves was measured in each region and statistically compared. In both the PGP- and CGRP-positive nerves, the nerve distribution density significantly increased vertically toward the mandibular canal from the alveolar crest and horizontally toward the periodontal ligament from the periosteum. From the CGRP- to PGP-positive nerve ratio, the pain-sensitive nerve accounted for approximately >70% in each region. Pain would therefore be more likely to develop when surgical invasiveness deepens toward the mandibular canal or periodontal ligament. Therefore, sufficient local anesthetic infiltration and/or combined use of conduction anesthesia or periodontal ligament injection may be required. These results may aid in the development of more effective surgical and anesthetic techniques for mandibular surgery.
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Péptido Relacionado con Gen de Calcitonina , Mandíbula , Proceso Alveolar , Animales , Inmunohistoquímica , Mandíbula/inervación , Ligamento Periodontal , Ratas , Ratas WistarRESUMEN
To assess the effect of epinephrine-containing local anesthetics on vasoconstriction, we immunohistochemically measured the intravascular lumen area in different regions of the mandible. Twelve male Wistar rats were used. General anesthesia was induced and maintained with sevoflurane. Infiltration anesthesia was performed with 0.2 mL of epinephrine-free 2% lidocaine (E-) near the left mandibular first molar and with 0.2 mL of epinephrine-containing 2% lidocaine (E+) near the right mandibular first molar. After decalcification, the specimens were paraffinized, and thin sections were prepared and immunohistologically stained with an antismooth muscle actin antibody. The intravascular lumen area was measured in the mucosa, periodontal membrane, Haversian/Volkmann's canal, and bone marrow. A Mann-Whitney U test was used for statistical processing, and p < .05 was considered to indicate a statistically significant difference. In the oral mucosa and the periodontal membrane, E+ had a significantly smaller vascular lumen area than E-. In the Haversian/Volkmann's canal and the bone marrow, no significant intergroup difference was observed in the intravascular lumen area. We postulate that this is due to a low smooth muscle content of blood vessels in the mandible and suggest that the vasoconstrictive effect of epinephrine-containing local anesthetics within the mandible is ineffective.
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Anestésicos Locales/administración & dosificación , Epinefrina/administración & dosificación , Lidocaína/administración & dosificación , Mandíbula/irrigación sanguínea , Músculo Liso Vascular/efectos de los fármacos , Vasoconstricción/efectos de los fármacos , Vasoconstrictores/administración & dosificación , Actinas/metabolismo , Animales , Biomarcadores/metabolismo , Inyecciones , Masculino , Músculo Liso Vascular/metabolismo , Ratas WistarRESUMEN
BACKGROUND: Epithelial-mesenchymal transition (EMT) is considered to play a critical role in cancer progression and metastasis. However, the impact of EMT on the prognosis of hepatocellular carcinoma (HCC) is still elusive. In this study, we examined the relationship between the expression of EMT markers and recurrence-free survival (RFS) and overall survival (OS) in HCC patients after hepatic resection. SUMMARY: The mRNA expression of 15 genes related to EMT was assessed by quantitative real-time polymerase chain reaction in cancerous tissues from 72 patients who underwent hepatic resection of HCC between January 2005 and December 2010 at our hospital. The upregulation of TWIST and the downregulation of tight junction protein ZO-1 (TJP1) were significantly associated with shorter RFS as well as OS. Increased levels of TWIST and decreased levels of TJP1 should be predictive markers for poor prognosis in patients with HCC after hepatectomy; those could serve as potential biomarkers for the treatment of HCC. Key Messages: A low level of TJP1 and high level of TWIST expression were prognostic factors predicting HCC after hepatic resection.
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Carcinoma Hepatocelular/cirugía , Neoplasias Hepáticas/cirugía , Proteínas Nucleares/análisis , Proteína 1 Relacionada con Twist/análisis , Proteína de la Zonula Occludens-1/análisis , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Línea Celular Tumoral , Movimiento Celular/genética , Transición Epitelial-Mesenquimal/genética , Femenino , Perfilación de la Expresión Génica , Hepatectomía/métodos , Humanos , Masculino , Persona de Mediana Edad , Pronóstico , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
The effect of motor cortex stimulation (MCS) therapy for deafferentation pain was evaluated based on c-Fos, a known pain marker. Nineteen mature cats weighing 1.5-3.5 kg were used. Cats were divided into three groups: a deafferentation pain group in which the left trigeminal ganglion was destroyed, an MCS group in which MCS was used following destruction of the trigeminal ganglion, and a control group. Sites and levels of c-Fos expression were examined immunohistochemically. The percentage of c-Fos-positive cells in the left spinal nucleus of the trigeminus, the bilateral insula, and the bilateral operculum increased in both the deafferentation pain and the MCS groups. There were no statistically significant differences between these groups. In the cingulate gyrus, the percentage of c-Fos-positive cells increased bilaterally in the deafferentation pain group and the MCS group, but the increase was greater in the MCS group. The increase in c-Fos-positive cells in the left spinal nucleus of the trigeminus in the deafferentation group may reflect reported electrical hyperactivity. The cingulate gyrus, insula, and parietal operculum were activated after deafferentation. This change (increase in c-Fos positive cells) is related to the development of deafferentation pain. Pain relief due to MCS is not dependent on the suppression of the activated left spinal nucleus of the trigeminus or the descending analgesic mechanism of the brain stem. Activation of the cingulate gyrus appears to be a factor in the analgesic mechanism of MCS.
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Causalgia/genética , Causalgia/terapia , Estimulación Encefálica Profunda , Modelos Animales de Enfermedad , Expresión Génica/genética , Corteza Motora/fisiopatología , Proteínas Proto-Oncogénicas c-fos/genética , Animales , Mapeo Encefálico , Gatos , Causalgia/fisiopatología , Dominancia Cerebral/genética , Dominancia Cerebral/fisiologíaRESUMEN
Chronic subdural hematoma (CSH) is a common disease that is treated with burr hole drainage by neurosurgeons. The outcome of CSH is mostly favorable. We treated 2 cases with bilateral occipital lobe infarction due to CSH. A 57-year-old woman was ambulatory when she visited a clinic for evaluation of headache. One hour after the CT was taken, she developed acute impairment of consciousness, so that she was referred to our hospital. The second patient was a 73-year-old woman with a history of depression who was involved in a traffic accident 5 weeks before admission to our hospital. She was at first admitted to a psychiatric hospital for evaluation of gait disturbance. Three weeks after she was admitted to the psychiatric hospital, she fell into a coma. She was referred to our hospital. Their brain CT on admission revealed compressed ambient and interpeduncular cistern and bilateral CSH. Although burr hole drainage surgery was performed, the 2 patients developed severe sequelae due to occipital lobe infarction caused by central transtentorial herniation.
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Hematoma Subdural Crónico/cirugía , Infarto/cirugía , Lóbulo Occipital/cirugía , Anciano , Femenino , Hematoma Subdural Crónico/etiología , Humanos , Infarto/complicaciones , Persona de Mediana Edad , Tomografía Computarizada por Rayos X/métodos , Resultado del TratamientoRESUMEN
PURPOSE: The involvement of the epithelial mesenchymal transition (EMT) in the process of corneal wound healing remains largely unclear. The purpose of the present study was to gain insight into Slug expression and corneal wound healing. METHODS: Slug expression during wound healing in the murine cornea was evaluated using fluorescence staining in vivo. Slug or Snail was stably introduced into human corneal epithelial cells (HCECs). These stable transfectants were evaluated for the induction of the EMT, cellular growth, migration activity, and expression changes in differentiation-related molecules. RESULTS: Slug, but not Snail, was clearly expressed in the nuclei of corneal epithelial cells in basal lesion of the corneal epithelium during wound healing in vivo. The overexpression of Slug or Snail induced an EMT-like cellular morphology and cadherin switching in HCECs, indicating that these transcription factors were able to mediate the typical EMT in HCECs. The overexpression of Slug or Snail suppressed cellular proliferation but enhanced the migration activity. Furthermore, ABCG2, TP63, and keratin 19, which are known as stemness-related molecules, were downregulated in these transfectants. CONCLUSIONS: It was found that Slug is upregulated during corneal wound healing in vivo. The overexpression of Slug mediated a change in the cellular phenotype affecting proliferation, migration, and expression levels of differentiation-related molecules. This is the first evidence that Slug is regulated during the process of corneal wound healing in the corneal epithelium in vivo, providing a novel insight into the EMT and Slug expression in corneal wound healing.
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Epitelio Corneal/patología , Lesiones Oculares/metabolismo , ARN Mensajero/genética , Factores de Transcripción/genética , Regulación hacia Arriba , Cicatrización de Heridas/fisiología , Animales , Western Blotting , Línea Celular , Movimiento Celular , Modelos Animales de Enfermedad , Epitelio Corneal/lesiones , Epitelio Corneal/metabolismo , Lesiones Oculares/patología , Humanos , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos BALB C , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia Snail , Factores de Transcripción/biosíntesisRESUMEN
SRPX2 (Sushi repeat-containing protein, X-linked 2) has recently emerged as a multifunctional protein that is involved in seizure disorders, angiogenesis and cellular adhesion. Here, we analyzed this protein biochemically. SRPX2 protein was secreted with a highly posttranslational modification. Chondroitinase ABC treatment completely decreased the molecular mass of purified SRPX2 protein to its predicted size, whereas heparitinase, keratanase and hyaluroinidase did not. Secreted SRPX2 protein was also detected using an anti-chondroitin sulfate antibody. These results indicate that SRPX2 is a novel chondroitin sulfate proteoglycan (CSPG). Furthermore, a binding assay revealed that hepatocyte growth factor dose-dependently binds to SRPX2 protein, and a ligand-glycosaminoglycans interaction was speculated to be likely in proteoglycans. Regarding its molecular architecture, SRPX2 has sushi repeat modules similar to four other CSPGs/lecticans; however, the molecular architecture of SRPX2 seems to be quite different from that of the lecticans. Taken together, we found that SRPX2 is a novel CSPG that is overexpressed in gastrointestinal cancer cells. Our findings provide key glycobiological insight into SRPX2 in cancer cells and demonstrate that SRPX2 is a new member of the cancer-related proteoglycan family.
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Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Neoplasias Gastrointestinales/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Línea Celular Tumoral , Condroitina ABC Liasa/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Gastrointestinales/genética , Regulación Neoplásica de la Expresión Génica , Glicosaminoglicanos/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Proteínas de la Membrana , Proteínas de Neoplasias , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Unión Proteica , Procesamiento Proteico-PostraduccionalRESUMEN
Resveratrol (3, 4', 5-trihydroxy-trans-stilbene), a natural phytoalexin found in grapes and wine, has anti-proliferative activity on human-derived cancer cells. In our study, we used a conventional condensation reaction between aldehydes and amines to provide a number of aza-resveratrol (3, 4', 5-trihydroxy-trans- aza-stilbene) derivatives in an attempt to screen for compounds with resveratrol's action but with increased potency. Aza-resveratrol and its hydroxylated derivative (3, 4, 4', 5-tetrahydroxy-trans- aza-stilbene) showed a more enhanced anti-proliferative effect than resveratrol in an MCF-7 breast carcinoma cell line. To identify the cellular targets of the aza derivatives of resveratrol, we conjugated the latter aza-stilbene compound with epoxy-activated agarose and performed affinity purification. Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, was identified as a major target protein in MCF-7 cell lysates using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). The aza-resveratrol and its hydroxylated derivative, but not resveratrol, were also found to be potent inhibitors of MIF tautomerase activity, which may be associated with their inhibitory effects on MIF bioactivity for cell growth.
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Compuestos Aza/farmacología , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Secuencia de Aminoácidos , Compuestos Aza/síntesis química , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Células MCF-7 , Datos de Secuencia Molecular , Resveratrol , Estilbenos/químicaRESUMEN
Whole genome-scale integrated analyses of exon array and array-comparative genomic hybridization are expected to enable the identification of unknown genetic features of cancer cells. Here, we evaluated this approach in 22 gastric and colorectal cancer cell lines, focusing on protein kinase genes and genes belonging to the cadherin-catenin family. Regarding alternative splicing patterns, several cancer cell lines predominantly expressed isoform 1 of protein kinase A catalytic subunit beta (PRKACB). Paired gastric cancer specimens demonstrated that isoform 1 of PRKACB was a novel cancer-related variant transcript in gastric cancers. In addition, the exon array analysis clearly identified exon 3 or exon 3-4 skipping in catenin beta 1, a short intron insertion with exon 9 skipping in CDH1, and a deletional transcript of CDH13. These abnormal transcripts were shown to have arisen from small genomic deletions. Meanwhile, an integrated analysis of 11 gastric cancer cell lines revealed that four cell lines amplified fibroblast growth factor receptor 2, with truncated forms observed in two of the cell lines. Gene amplification, and not the truncated form, was found to determine the sensitivity to a fibroblast growth factor receptor inhibitor, indicating that our cell line panel might be useful for cell-based evaluations of specific inhibitors. Using an integrated analysis, we identified several abnormal transcripts and genomic alterations in gastric and colorectal cancer cells. Our approach might enable genetic changes to be identified more efficiently, and the present results warrant further investigation using clinical samples and integrated analyses.
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Cadherinas/genética , Cateninas/genética , Neoplasias Colorrectales/genética , Proteínas Quinasas/genética , Neoplasias Gástricas/genética , Empalme Alternativo , Secuencia de Bases , Línea Celular Tumoral , Hibridación Genómica Comparativa , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Exones , Amplificación de Genes , Dosificación de Gen , Genómica , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Isoformas de Proteínas/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Análisis de Secuencia de ADNRESUMEN
Acquired resistance to antiangiogenic drugs has emerged as a potentially important issue in clinical settings; however, the underlying molecular and cellular mechanism of resistance to vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase inhibitor (TKI) remains largely unclear. We evaluated the cellular characteristics of human umbilical vein endothelial cell (HUVEC) clones, which are resistant to VEGFR2-TKI (Ki8751) to elucidate this mechanism of resistance to antiangiogenic drugs. Resistant HUVEC clones were 10-fold more resistant to VEGFR2-TKI than the parental cells and they exhibited an almost complete absence of VEGF-mediated cellular proliferation. The mRNA expression analysis revealed that expression of VEGFR1, VEGFR2 and VEGFR3 was lower in resistant clones, while that of several angiogenic ligands was increased. The protein expression of VEGFR2 was markedly down-regulated in two (R5 and R6 clone) out of five resistant clones. Focusing on the R5 clone, VEGF stimulation did not increase the phosphorylation of VEGFR2 or the dimerization of VEGFR2. The inhibition of phospho-AKT by VEGFR2-TKI was also weakened more than 10-fold in the R5 clone. Finally, a microarray analysis revealed that some angiogenesis-associated, and some angiogenesis-specific genes, including platelet endothelial cell adhesion molecule 1 (PECAM1)/CD31, homeobox A9 (HOXA9), and endothelial cell-specific molecule 1 (ESM1), were remarkably down-regulated in all the resistant clones compared with the parental cells. HUVEC clones resistant to VEGFR2-TKI exhibited down-regulation of VEGFR2, a decreased signal response to VEGF stimulation, and the loss of vascular endothelial markers. These results strongly suggest that an escape from VEGFR2 signaling-dependency is one of the cellular mechanisms of resistance to VEGFR2-TKI in vascular endothelial cells.
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Endotelio Vascular/efectos de los fármacos , Compuestos de Fenilurea/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinolinas/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Secuencia de Bases , Línea Celular Transformada , Cartilla de ADN , Resistencia a Medicamentos , Endotelio Vascular/citología , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We describe a 19-year-old woman with onset of epileptic seizure, and a small mural nodule and multicystic lesions with severe brain edema located in the right frontal lobe. At surgery, the tumor and a clear margin was removed, and symptoms improved postoperatively. Extended local radiotherapy (60 Gy) was performed. Histopathological examination revealed oligodendroglioma-like tumor cells with a perinuclear halo. The tumor cells extended processes toward CD34-positive proliferating vessels, which resemble a basement membrane. These proliferating vessels formed a tumor membrane so that there was a clear margin between the tumor and brain tissue. Tumor cells were positive for epithelial membrane antigen in a dot-like pattern. MIB-1 staining index was 50.6%. Electron microscopy showed cilia and zipper-like junctions, and anaplastic clear-cell ependymoma grade III was diagnosed. A characteristic of the case was formation of a tumor membrane by proliferating tumor blood vessels. Fluorescence in situ hybridization showed 1p/19q deletions, and the concentration of erythropoietin in the cyst fluid was abnormally high, at 1,859.4 mIU/ml. Erythropoietin and erythropoietin receptors were verified with immunohistochemical staining.
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Neoplasias Encefálicas/patología , Deleción Cromosómica , Cromosomas Humanos Par 19 , Cromosomas Humanos Par 1 , Ependimoma/patología , Eritropoyetina/análisis , Adulto , Neoplasias Encefálicas/química , Neoplasias Encefálicas/genética , Ependimoma/química , Ependimoma/genética , Femenino , HumanosRESUMEN
The epithelial mesenchymal transition (EMT) has emerged as a pivotal event in the development of the invasive and metastatic potentials of cancer progression. Sorafenib, a VEGFR inhibitor with activity against RAF kinase, is active against hepatocellular carcinoma (HCC); however, the possible involvement of sorafenib in the EMT remains unclear. Here, we examined the effect of sorafenib on the EMT. Hepatocyte growth factor (HGF) induced EMT-like morphologic changes and the upregulation of SNAI1 and N-cadherin expression. The downregulation of E-cadherin expression in HepG2 and Huh7 HCC cell lines shows that HGF mediates the EMT in HCC. The knockdown of SNAI1 using siRNA canceled the HGF-mediated morphologic changes and cadherin switching, indicating that SNAI1 is required for the HGF-mediated EMT in HCC. Interestingly, sorafenib and the MEK inhibitor U0126 markedly inhibited the HGF-induced morphologic changes, SNAI1 upregulation, and cadherin switching, whereas the PI3 kinase inhibitor wortmannin did not. Collectively, these findings indicate that sorafenib downregulates SNAI1 expression by inhibiting mitogen-activated protein kinase (MAPK) signaling, thereby inhibiting the EMT in HCC cells. In fact, a wound healing and migration assay revealed that sorafenib completely canceled the HGF-mediated cellular migration in HCC cells. In conclusion, we found that sorafenib exerts a potent inhibitory activity against the EMT by inhibiting MAPK signaling and SNAI1 expression in HCC. Our findings may provide a novel insight into the anti-EMT effect of tyrosine kinase inhibitors in cancer cells.
Asunto(s)
Bencenosulfonatos/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Neoplasias Hepáticas/tratamiento farmacológico , Piridinas/farmacología , Antineoplásicos/farmacología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Regulación hacia Abajo , Transición Epitelial-Mesenquimal/efectos de los fármacos , Factor de Crecimiento de Hepatocito/genética , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Niacinamida/análogos & derivados , Compuestos de Fenilurea , Transducción de Señal , Sorafenib , TransfecciónRESUMEN
PURPOSE: The aim of this study was to investigate the expression changes of epithelial mesenchymal transition (EMT)-related molecules induced by TGF-ß signaling in a human corneal epithelial cell line (HCECs). METHODS: The cellular response to TGF-ß was evaluated by immunoblotting, quantitative real-time RT-PCR, and immunofluorescence microscopy in HCECs. RESULTS: TGF-ß significantly increased mRNA expression of SNAI1, SNAI2, VIM, and FN1, but not TWIST1 through Smad and non-Smad pathways in HCECs. Protein expression of a mesenchymal marker N-cadherin was dose-dependently increased and that of an epithelial marker of E-cadherin was decreased by TGF-ß. TGF-ß, but not EGF, mediated the EMT-like morphologic changes. Both TGF-ß and EGF were capable of upregulating SNAI1 and SNAI2 by about two-fold within a short response time. However, a detailed time course analysis revealed drastically different expression patterns, with TGF-ß mediating a sustained upregulation of SNAI1 and SNAI2 for at least for 6 days and EGF allowing a return to the baseline expression values after 8 â¼ 12 h. These data indicate that TGF-ß, but not EGF, induces sustained upregulation of SNAI1 and SNAI2 in HCECs. CONCLUSIONS: TGF-ß induces sustained upregulation of SNAI1 and SNAI2 through Smad and non-Smad pathways, EMT-like morphologic changes, downregulation of E-cadherin, and upregulation of N-cadherin in HCECs. The authors' findings provide insight into the TGF-ß signaling and the temporal expression patterns of EMT-inducible transcription factors in HCECs.
Asunto(s)
Epitelio Corneal/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Smad/metabolismo , Factores de Transcripción/genética , Factor de Crecimiento Transformador beta/farmacología , Proteínas de Unión al GTP rho/metabolismo , Western Blotting , Cadherinas/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Epitelio Corneal/citología , Humanos , Immunoblotting , Mesodermo/citología , Microscopía Fluorescente , Microscopía de Contraste de Fase , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción de la Familia SnailRESUMEN
PURPOSE: BIBF 1120 is a potent, orally available triple angiokinase inhibitor that inhibits VEGF receptors (VEGFR) 1, 2, and 3, fibroblast growth factor receptors, and platelet-derived growth factor receptors. This study examined the antitumor effects of BIBF 1120 on hepatocellular carcinoma (HCC) and attempted to identify a pharmacodynamic biomarker for use in early clinical trials. EXPERIMENTAL DESIGN: We evaluated the antitumor and antiangiogenic effects of BIBF 1120 against HCC cell line both in vitro and in vivo. For the pharmacodynamic study, the phosphorylation levels of VEGFR2 in VEGF-stimulated peripheral blood leukocytes (PBL) were evaluated in mice inoculated with HCC cells and treated with BIBF 1120. RESULTS: BIBF 1120 (0.01 µmol/L) clearly inhibited the VEGFR2 signaling in vitro. The direct growth inhibitory effects of BIBF 1120 on four HCC cell lines were relatively mild in vitro (IC(50) values: 2-5 µmol/L); however, the oral administration of BIBF 1120 (50 or 100 mg/kg/d) significantly inhibited the tumor growth and angiogenesis in a HepG2 xenograft model. A flow cytometric analysis revealed that BIBF 1120 significantly decreased the phosphotyrosine (pTyr) levels of VEGFR2(+)CD45(dim) PBLs and the percentage of VEGFR2(+)pTyr(+) PBLs in vivo; the latter parameter seemed to be a more feasible pharmacodynamic biomarker. CONCLUSIONS: We found that BIBF 1120 exhibited potent antitumor and antiangiogenic activity against HCC and identified VEGFR2(+)pTyr(+) PBLs as a feasible and noninvasive pharmacodynamic biomarker in vivo.
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Inhibidores de la Angiogénesis/farmacología , Antineoplásicos/farmacología , Carcinoma Hepatocelular/genética , Indoles/farmacología , Leucocitos/metabolismo , Neoplasias Hepáticas/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/biosíntesis , Animales , Biomarcadores/metabolismo , Carcinoma Hepatocelular/tratamiento farmacológico , Línea Celular Tumoral , Proliferación Celular , Femenino , Citometría de Flujo/métodos , Humanos , Concentración 50 Inhibidora , Neoplasias Hepáticas/tratamiento farmacológico , Ratones , Ratones Desnudos , Trasplante de NeoplasiasRESUMEN
OBJECTIVE: The purpose of this study was to assess the usefulness of post-vascular phase (PVP) images of contrast-enhanced ultrasonography (CE-US) in the evaluation of the gross types of hepatocellular carcinoma (HCC) that is closely related to the malignant potential of the tumor. METHODS: A total of 29 patients with 40 HCCs of <5 cm in diameter, who underwent hepatic resection, were enrolled. The gross type of the tumor was evaluated using real-time scanning during the PVP of CE-US with Sonazoid prior to surgery. The tumors were classified into three types based on the macroscopic classification of the Liver Cancer Study Group of Japan: single nodular (SN) type, single nodular with extranodular growth (SNEG) type, and confluent multinodular (CMN) type. The ability of CE-US to correctly depict the gross type of HCC was evaluated. RESULTS: 26 tumors were macroscopically diagnosed as the SN type, 11 tumors as the SNEG type, and 3 tumors as the CMN type. The sensitivity, specificity and accuracy of CE-US were 96, 80 and 90%, respectively. CONCLUSION: The PVP image of CE-US with Sonazoid is a useful tool in the evaluation of the gross type of HCC and is considered essential in deciding treatment strategy.
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Carcinoma Hepatocelular/diagnóstico por imagen , Medios de Contraste , Compuestos Férricos , Hierro , Neoplasias Hepáticas/diagnóstico por imagen , Óxidos , Anciano , Carcinoma Hepatocelular/cirugía , Femenino , Humanos , Neoplasias Hepáticas/cirugía , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , UltrasonografíaRESUMEN
Bortezomib, a selective 26S proteasome inhibitor, has shown clinical benefits against refractory multiple myeloma. The indirect anti-angiogenic activity of bortezomib has been widely recognized; however, the growth-inhibitory mechanism of bortezomib on vascular endothelial cells remains unclear, especially on the cell cycle. Here, we showed that bortezomib (2 nM of the IC(50) value) potently inhibited the cellular growth of human umbilical vascular endothelial cells (HUVECs) via a vascular endothelial growth factor receptor (VEGFR)-independent mechanism resulting in the induction of apoptosis. Bortezomib significantly increased the vascular permeability of HUVECs, whereas a VEGFR-2 tyrosine kinase inhibitor decreased it. Interestingly, a cell cycle analysis using flow cytometry, the immunostaining of phospho-histone H3, and Giemsa staining revealed that bortezomib suppressed the G2/M transition of HUVECs, whereas the mitotic inhibitor paclitaxel induced M-phase accumulation. A further analysis of cell cycle-related proteins revealed that bortezomib increased the expression levels of cyclin B1, the cdc2/cyclin B complex, and the phosphorylation of all T14, Y15, and T161 residues on cdc2. Bortezomib also increased the ubiquitination of cyclin B1 and wee1, but inhibited the kinase activity of the cdc2/cyclin B complex. These protein modifications support the concept that bortezomib suppresses the G2/M transition, rather than causing M-phase arrest. In conclusion, we demonstrated that bortezomib potently inhibits cell growth by suppressing the G2/M transition, modifying G2/M-phase-related cycle regulators, and increasing the vascular permeability of vascular endothelial cells. Our findings reveal a cell cycle-related mode of action and strongly suggest that bortezomib exerts an additional unique vascular disrupting effect as a vascular targeting drug.
Asunto(s)
Antineoplásicos/farmacología , Ácidos Borónicos/farmacología , División Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Fase G2/efectos de los fármacos , Inhibidores de Proteasoma , Pirazinas/farmacología , Apoptosis/efectos de los fármacos , Bortezomib , Proteína Quinasa CDC2 , Permeabilidad Capilar/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Ciclina B/antagonistas & inhibidores , Quinasas Ciclina-Dependientes , Células Endoteliales/fisiología , Humanos , Complejo de la Endopetidasa Proteasomal , Factor A de Crecimiento Endotelial Vascular/fisiologíaRESUMEN
Forkhead box Q1 (FOXQ1) is a member of the forkhead transcription factor family, and it has recently been proposed to participate in gastric acid secretion and mucin gene expression in mice. However, the role of FOXQ1 in humans and especially in cancer cells remains unknown. We found that FOXQ1 mRNA is overexpressed in clinical specimens of colorectal cancer (CRC; 28-fold/colonic mucosa). A microarray analysis revealed that the knockdown of FOXQ1 using small interfering RNA resulted in a decrease in p21(CIP1/WAF1) expression, and a reporter assay and a chromatin immunoprecipitation assay showed that p21 was one of the target genes of FOXQ1. Stable FOXQ1-overexpressing cells (H1299/FOXQ1) exhibited elevated levels of p21 expression and inhibition of apoptosis induced by doxorubicin or camptothecin. Although cellular proliferation was decreased in H1299/FOXQ1 cells in vitro, H1299/FOXQ1 cells significantly increased tumorigenicity [enhanced green fluorescent protein (EGFP): 2/15, FOXQ1: 7/15] and enhanced tumor growth (437 +/- 301 versus 1735 +/- 769 mm3, P < 0.001) in vivo. Meanwhile, stable p21 knockdown of H1299/FOXQ1 cells increased tumor growth, suggesting that FOXQ1 promotes tumor growth independent of p21. Microarray analysis of H1299/EGFP and H1299/FOXQ1 revealed that FOXQ1 overexpression upregulated several genes that have positive roles for tumor growth, including VEGFA, WNT3A, RSPO2, and BCL11A. CD31 and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling staining of the tumor specimens showed that FOXQ1 overexpression mediated the angiogenic and antiapoptotic effect in vivo. In conclusion, FOXQ1 is overexpressed in CRC and enhances tumorigenicity and tumor growth presumably through its angiogenic and antiapoptotic effects. Our findings show that FOXQ1 is a new member of the cancer-related FOX family.
Asunto(s)
Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Factores de Transcripción Forkhead/biosíntesis , Animales , Apoptosis/genética , Procesos de Crecimiento Celular/fisiología , Línea Celular Tumoral , Neoplasias Colorrectales/irrigación sanguínea , Neoplasias Colorrectales/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Femenino , Factores de Transcripción Forkhead/genética , Expresión Génica , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Activación Transcripcional , Transfección , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The underlying mechanism regulating the expression of the cancer stem cell/tumor-initiating cell marker CD133/prominin-1 in cancer cells remains largely unclear, although knowledge of this mechanism would likely provide important biological information regarding cancer stem cells. Here, we found that the inhibition of mTOR signaling up-regulated CD133 expression at both the mRNA and protein levels in a CD133-overexpressing cancer cell line. This effect was canceled by a rapamycin-competitor, tacrolimus, and was not modified by conventional cytotoxic drugs. We hypothesized that hypoxia-inducible factor-1 alpha (HIF-1 alpha), a downstream molecule in the mTOR signaling pathway, might regulate CD133 expression; we therefore investigated the relation between CD133 and HIF-1 alpha. Hypoxic conditions up-regulated HIF-1 alpha expression and inversely down-regulated CD133 expression at both the mRNA and protein levels. Similarly, the HIF-1 alpha activator deferoxamine mesylate dose-dependently down-regulated CD133 expression, consistent with the effects of hypoxic conditions. Finally, the correlations between CD133 and the expressions of HIF-1 alpha and HIF-1 beta were examined using clinical gastric cancer samples. A strong inverse correlation (r = -0.68) was observed between CD133 and HIF-1 alpha, but not between CD133 and HIF-1 beta. In conclusion, these results indicate that HIF-1 alpha down-regulates CD133 expression and suggest that mTOR signaling is involved in the expression of CD133 in cancer cells. Our findings provide a novel insight into the regulatory mechanisms of CD133 expression via mTOR signaling and HIF-1 alpha in cancer cells and might lead to insights into the involvement of the mTOR signal and oxygen-sensitive intracellular pathways in the maintenance of stemness in cancer stem cells.
Asunto(s)
Antígenos CD/biosíntesis , Glicoproteínas/biosíntesis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Neoplasias/metabolismo , Proteínas Quinasas/metabolismo , Antígeno AC133 , Antígenos CD/genética , Línea Celular Tumoral , Cromonas/farmacología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Glicoproteínas/genética , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/biosíntesis , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Morfolinas/farmacología , Neoplasias/genética , Péptidos/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Transducción de Señal , Sirolimus/farmacología , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Serina-Treonina Quinasas TOR , Tacrolimus/farmacología , Transcripción Genética , Regulación hacia Arriba/genéticaRESUMEN
BACKGROUND: DelE746_A750-type EGFR is a constitutively active type of mutation that enhances EGFR signaling. However, the changes in gene expression that occur in mutant EGFR-harboring cells has not been fully studied. MATERIALS AND METHODS: A gene expression analysis of HEK293 cells transfected with wild-type or mutant EGFR was performed focusing on the significant gene. RESULTS: Early growth response 1 (EGR1), a transcription factor, was the most strongly up-regulated gene in mutant EGFR-transfected cells among the genes examined. An increase in EGR1 expression in the mutant EGFR cells was confirmed using RT-PCR or immunoblotting. The expression was up-regulated by EGF stimulation and down-regulated by EGFR-tyrosine kinase inhibitor. In addition, the MEK inhibitor U0126 inhibited EGR1 expression, while the phosphatidylinositol 3-kinase inhibitor LY294002 did not. CONCLUSION: Mutant EGFR constitutively up-regulates EGR1 through the ERK pathway, and its expression is correlated with EGFR signal activation. Findings provide an insight into a target gene of mutant EGFR and further improve the understanding of the oncogenic properties of EGFR.
Asunto(s)
Proteína 1 de la Respuesta de Crecimiento Precoz/metabolismo , Receptores ErbB/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación/genética , Transducción de Señal , Células Cultivadas , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Humanos , Immunoblotting , Riñón/citología , Riñón/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Constitutively active mutations of epidermal growth factor receptor (EGFR) (delE746_A750) activate downstream signals, such as ERK and Akt, through the phosphorylation of tyrosine residues in the C-terminal region of EGFR. These pathways are thought to be important for cellular sensitivity to EGFR tyrosine kinase inhibitors (TKI). To examine the correlation between phosphorylation of the tyrosine residues in the C-terminal region of EGFR and cellular sensitivity to EGFR TKI, we used wild-type (wt) EGFR, as well as the following constructs: delE746_A750 EGFR; delE746_A750 EGFR with substitution of seven tyrosine residues to phenylalanine in the C-terminal region; and delE746_A750 EGFR with a C-terminal truncation at amino acid 980. These constructs were transfected stably into HEK293 cells and designated HEK293/Wt, HEK293/D, HEK293/D7F, and HEK293/D-Tr, respectively. The HEK293/D cells were found to be 100-fold more sensitive to EGFR TKI (AG1478) than HEK293/Wt. Surprisingly, the HEK293/D7F and HEK293/D-Tr cells, transfected with EGFR lacking the C-terminal autophosphorylation sites, retained high sensitivity to EGFR TKI. In these three high-sensitivity cells, the ERK pathway was activated without ligand stimulation, which was inhibited by EGFR TKI. In addition, although EGFR in the HEK293/D7F and HEK293/D-Tr cells lacked significant tyrosine residues for EGFR signal transduction, phosphorylation of Src homology and collagen homology (Shc) was spontaneously activated in these cells. Our results indicate that tyrosine residues in the C-terminal region of EGFR are not required for cellular sensitivity to EGFR TKI, and that an as-yet-unknown signaling pathway of EGFR may exist that is independent of the C-terminal region of EGFR.