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BACKGROUND: Large non-apoptotic vesicles released from the plasma membrane protrusions are classified as large-EVs (LEVs). However, the triggers of LEV secretion and their functions in tumors remain unknown. METHODS: Coculture system of cancer cells, peritoneal mesothelial cells (PMCs), and macrophages (MΦs) was conducted to observe cell-cell contact-mediated LEV secretion. Lineage tracing of PMCs was performed using Wt1CreERT2-tdTnu mice to explore the effects of LEVs on PMCs in vivo, and lymphangiogenesis was assessed by qRT-PCR and flow-cytometry. RESULTS: In peritoneal dissemination, cancer cells expressing Ephrin-B (EFNB) secreted LEVs upon the contact with PMCs expressing ephrin type-B (EphB) receptors, which degraded mesothelial barrier by augmenting mesothelial-mesenchymal transition. LEVs were incorporated in subpleural MΦs, and these MΦs transdifferentiated into lymphatic endothelial cells (LEC) and integrated into the lymphatic vessels. LEC differentiation was also induced in PMCs by interacting with LEV-treated MΦs, which promoted lymphangiogenesis. Mechanistically, activation of RhoA-ROCK pathway through EFNB reverse signaling induced LEV secretion. EFNBs on LEVs activated EphB forward signaling in PMC and MΦs, activating Akt, ERK and TGF-ß1 pathway, which were indispensable for causing MMT and LEC differentiation. LEVs accelerated peritoneal dissemination and lymphatic invasions by cancer cells. Blocking of EFNBs on LEVs using EphB-Fc-fusion protein attenuated these events. CONCLUSIONS: EFNBhigh cancer cells scattered LEVs when they attached to PMCs, which augmented the local reactions of PMC and MΦ (MMT and lymphangiogenesis) and exaggerated peritoneal dissemination.
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PURPOSE: IDH-wildtype (IDHwt) diffuse gliomas are treated as glioblastoma, however, some of these may show less aggressive clinical courses. The authors investigated the clinical, histopathological, and molecular characteristics of such IDHwt indolent diffuse gliomas (iDGwt), which have not been well documented in the literature. METHODS: Adult patients with IDHwt gliomas admitted between 2011 and 2020 were surveyed. In this particular study, the clinical indolence was defined mainly as having a small enhancing lesion and a stable period for more than 1 month before surgery. The current WHO diagnostic criteria were adapted for the diagnoses. Gene mutations and copy number changes in 43 representative glioma-associated genes, MGMT promoter methylation status, and survival data were compared with those of The Cancer Genome Atlas reference cohort. RESULTS: Nine out of 180 surveyed cases (5.0%) fulfilled the present criteria of the iDGwt. Considering the representative regulatory pathways, 8 (88.9%), 4 (44.4%), and 1 (11.1%) case had genetic alterations in the PI3K/MAPK, TP53, and RB pathways, respectively. The frequency of the RB pathway alteration was significantly lower than that in the reference cohort (281 of 362 cases: 77.6%). Two cases (22.2%) showing EGFR amplification met the diagnostic criteria for glioblastoma, and the frequency was significantly lower than that in the reference cohort (412 of 426 cases: 96.7%). The overall survival (median: 37.5 months) in the present series was significantly longer than that in the reference cohort (n = 426, median: 13.9 months). CONCLUSIONS: iDGwt lacked the molecular features of glioblastoma except for the PI3K/MAPK pathway alteration.
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Neoplasias Encefálicas , Glioblastoma , Glioma , Adulto , Neoplasias Encefálicas/genética , Glioblastoma/genética , Glioma/genética , Humanos , Isocitrato Deshidrogenasa/genética , Estimación de Kaplan-Meier , Mutación , Fosfatidilinositol 3-Quinasas , PronósticoRESUMEN
PURPOSE: To establish whether Sphingosine-1-phosphate (S1P) and sphingosine kinase 1 (SphK1) contribute to lymph node metastasis in esophageal squamous cell carcinoma. METHODS: Immunohistochemical analysis of SphK1 expression was performed using a tissue microarray containing 177 thoracic squamous cell esophageal cancer specimens resected at surgery, to investigate the association between intratumoral SphK1 expression and lymph node metastasis. Serum S1P levels and intratumoral SphK1 mRNA and protein expression were also evaluated in mice with vs. mice without lymph node metastasis in a murine lymph node metastasis model. RESULTS: Among 177 esophageal cancer patients, 127 (72%) were defined as being SphK1-positive. In univariate and multivariate analyses, SphK1 expression status was a significant factor contributing to lymph node metastasis and poorer 5-year overall survival. In the murine lymph node metastasis model, there was no difference in tumor volume or weight between the lymph node metastasis-negative and lymph node metastasis-positive groups. However, levels of SphK1 mRNA and protein and serum S1P levels were all much higher in the metastasis-positive group. CONCLUSIONS: S1P/SphK1 may be novel targets for inhibiting lymph node metastasis in esophageal squamous cell carcinoma, and may provide the basis for a therapeutic strategy to suppress lymph node metastasis.
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Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Expresión Génica , Lisofosfolípidos/genética , Lisofosfolípidos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Esfingosina/análogos & derivados , Anciano , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Metástasis Linfática , Lisofosfolípidos/sangre , Masculino , Ratones , Persona de Mediana Edad , Terapia Molecular Dirigida , ARN Mensajero/metabolismo , Esfingosina/sangre , Esfingosina/genética , Esfingosina/metabolismoRESUMEN
Cadmium is a toxic pollutant with occupational and environmental significance, due to its diverse toxic effects. Supramolecules that conjugate and decontaminate toxic metals have potential for use in treatment of cadmium intoxication. In addition, metal-coordinating ability has been postulated to contribute to the cytotoxic effects of anti-tumor agents such as cisplatin or bleomycin. Thiacalixarenes, cyclic oligomers of p-alkylphenol bridged by sulfur atoms, are supramolecules known to have potent coordinating ability to metal ions. In this study, we show that cadmium-coordinated thiacalix[4]arene tetrasulfate (TC4ATS-Cd) exhibits an anti-proliferative effect against T-cell leukemia cells. Cadmium exhibited cytotoxicity with IC50 values ranging from 36 to 129 µM against epithelia-derived cancer cell lines, while TC4ATS-Cd elicited no significant cytotoxicity (IC50 > 947 µM). However, a number of T-cell leukemia cell lines exhibited marked sensitivity to TC4ATS-Cd. In Jurkat cells, toxicity of TC4ATS-Cd occurred with an IC50 of 6.9 µM, which is comparable to that of 6.5 µM observed for cadmium alone. TC4ATS-Cd induced apoptotic cell death through activation of caspase-3 in Jurkat cells. In a xenograft model, TC4ATS-Cd (13 mg/kg) treatment significantly suppressed the tumor growth of Jurkat cells in mice. In addition, TC4ATS-Cd-treated mice exhibited significantly less cadmium accumulation in liver and kidney compared to equimolar cadmium-treated mice. These results suggest that cadmium-coordinated supramolecules may have therapeutic potential for treatment of T-cell leukemia.
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Compuestos de Cadmio/farmacología , Leucemia de Células T/tratamiento farmacológico , Fenoles/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Cadmio/farmacocinética , Cadmio/toxicidad , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Femenino , Humanos , Concentración 50 Inhibidora , Células Jurkat/efectos de los fármacos , Leucemia de Células T/patología , Ratones SCID , Fenoles/química , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Identification of the key molecules that mediate susceptibility to anticancer treatments would be highly desirable. Based on clinical and cell biological studies, we recently proposed that regenerating gene (REG) Iα may be such a molecule. In the present study, we hypothesized that REG Iα increases radiosensitivity through activation of mitogen-activated protein kinase (MAPK) pathways. To test that idea, we transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Iα and examined its involvement in MAPK signaling and its effect on susceptibility to radiotherapy. We found that REG Iα-expressing cells showed increased expression of c-Jun messenger RNA (mRNA) and phospho-c-Jun protein mediated via the c-Jun N-terminal kinase (JNK) pathway and extracellular signal-regulated kinase (ERK) pathway, as well as increased radiosensitivity. Immunohistochemical analysis confirmed the activation of c-Jun in tumors expressing REG Iα. Collectively, these findings suggest that REG Iα activates c-Jun via the JNK and ERK pathway, thereby enhancing radiosensitivity.
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Carcinoma de Células Escamosas/genética , Neoplasias Esofágicas/genética , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Litostatina/genética , Proteínas Proto-Oncogénicas c-jun/genética , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/radioterapia , Línea Celular Tumoral , Neoplasias Esofágicas/patología , Neoplasias Esofágicas/radioterapia , Carcinoma de Células Escamosas de Esófago , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/biosíntesis , Litostatina/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Proto-Oncogénicas c-jun/biosíntesis , ARN Mensajero/biosíntesis , Tolerancia a Radiación/genéticaRESUMEN
Mucin-type O-glycosylation is initiated by a large number of UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (GalNAc-T). Although extensive in vitro studies using synthetic peptides as substrates suggest that most GalNAc-Ts exhibit overlapping substrate specificities, many studies have shown that individual GalNAc-Ts play an important role in both animals and humans. Further investigations of the functions of individual GalNAc-Ts including in vivo substrate proteins and O-glycosylation sites are necessary. In this study, we attempted to generate single-chain variable fragment (scFv) antibodies to bind to GalNAc-T1, T2, T3, and T4 using a yeast two-hybrid system for screening a naive chicken scFv library. Several different scFvs were isolated against a single target GalNAc-T isoform specifically under expressed in yeast and were confirmed to be expressed in mammalian cells and to retain binding activity inside the cells. Generation of these specific antibodies provides an opportunity to modify and exploit antibodies for specific applications in investigations of GalNAc-T functions.
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N-Acetilgalactosaminiltransferasas/inmunología , Péptidos/inmunología , Anticuerpos de Cadena Única/biosíntesis , Técnicas del Sistema de Dos Híbridos , Secuencia de Aminoácidos , Animales , Línea Celular , Pollos , Humanos , Datos de Secuencia Molecular , Biblioteca de Péptidos , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/genéticaRESUMEN
Glechoma hederacea L. (Labiatae) has been used in folk medicine to treat various ailments for centuries. We investigated the effects of G. hederacea extract on melanogenesis in B16 melanoma cells. It significantly reduced both the cellular melanin content and tyrosinase activity in a concentration-dependent manner. An MTT assay did not reveal any obvious cytotoxicity. Furthermore, we found that G. hederacea extract decreased tyrosinase and microphthalmia-associated transcription factor protein expression, but did not inhibit tyrosinase-related protein-1 and tyrosinase-related protein-2 expression. RT-PCR analysis indicated that the antimelanogenic effect of G. hederacea extract might be due to inhibition of tyrosinase gene transcription. Moreover, this effect is regulated via suppression of microphthalmia-associated transcription factor protein expression. Our data indicate that G. hederacea extract inhibits melanin synthesis in B16 melanoma cells but is not cytotoxic. Hence it might prove a useful therapeutic agent for treating hyperpigmentation and an effective component of whitening cosmetics.
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Lamiaceae/química , Melaninas/biosíntesis , Melanoma Experimental/patología , Extractos Vegetales/farmacología , Agaricales/enzimología , Animales , Proliferación Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Ratones , Factor de Transcripción Asociado a Microftalmía/genética , Monofenol Monooxigenasa/antagonistas & inhibidoresRESUMEN
BACKGROUND: The mechanism underlying cancer cell metastasis from the tumor to regional lymph nodes is not yet fully understood. We hypothesized that peritumoral neutrophil accumulation promotes regional lymph node metastasis in thoracic esophageal squamous cell cancer. METHODS: Between 2010 and 2019, 126 thoracic esophageal squamous cell cancer patients received curative (R0) esophagectomy without preoperative treatment in our hospital. Using paraffin-embedded resected tumors, we performed immunohistochemical analysis of CD16b-positive neutrophil accumulation in the peritumoral area, which was defined as a 1-mm region centered on the border separating the malignant cell nests from the host tissue. The relationship between the density of peritumoral CD16b staining and pathological lymph node metastasis or 5-year overall survival was evaluated. RESULTS: Although the clinicopathological characteristics of CD16b-high and CD16b-low patients did not differ, greater pathological lymph node metastasis (P < .001) and lymphatic invasion by the tumor (P = .024) and a poorer 5-year survival (P = .010) were seen in CD16b-high patients. Moreover, CD16b-positive neutrophil density was generally higher in the peritumoral area than within the tumor itself. Univariate and multivariate analyses showed that CD16b-positive neutrophil accumulation was an independent factor for lymph node metastasis with an odds ratio >25 (P < .001). On the other hand, blood neutrophil counts did not correlate with lymph node metastasis. CONCLUSION: Peritumoral accumulation of CD16b-positive neutrophils is an independent factor strongly correlated with lymph node metastasis in thoracic esophageal squamous cell cancer.
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Carcinoma de Células Escamosas , Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , Células Epiteliales/patología , Carcinoma de Células Escamosas de Esófago/patología , Carcinoma de Células Escamosas de Esófago/cirugía , Esofagectomía , Humanos , Escisión del Ganglio Linfático , Ganglios Linfáticos/patología , Metástasis Linfática/patología , Estadificación de Neoplasias , Neutrófilos , Estudios RetrospectivosRESUMEN
Zinc is one of the trace elements which induce the proliferation and the differentiation of the osteoblast. In the previous study, we found that zinc ions (Zn(2+) ion)-releasing titanium implants had excellent bone fixation using a rabbit femurs model. In this study, we isolated the Zn(2+) ions (eluted Zn(2+) ion; EZ) released from the implant surface, and evaluated the effect of EZ on the osteogenesis of human bone marrow-derived mesenchymal cells (hBMCs). In the result, it was found that the EZ stimulated cell viability, osteoblast marker gene (type I collagen, osteocalcin (OC), alkaline phosphatase (ALP) and bone sialoprotein (BSP)) expressions and calcium deposition in hBMCs.
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Regeneración Ósea/efectos de los fármacos , Osteogénesis/efectos de los fármacos , Zinc/metabolismo , Fosfatasa Alcalina/biosíntesis , Animales , Células de la Médula Ósea/efectos de los fármacos , Cationes Bivalentes/metabolismo , Cationes Bivalentes/farmacología , Células Cultivadas , Colágeno Tipo I/biosíntesis , Implantes de Medicamentos/metabolismo , Implantes de Medicamentos/farmacología , Fémur , Sialoproteína de Unión a Integrina/biosíntesis , Mesodermo/efectos de los fármacos , Osteocalcina/biosíntesis , ARN Mensajero/biosíntesis , Conejos , Propiedades de Superficie , Titanio , Zinc/farmacologíaRESUMEN
Identification of reliable markers of radiosensitivity and the key molecules that enhance the susceptibility of esophageal cancer cells to anticancer treatments would be highly desirable. To identify molecules that confer radiosensitivity to esophageal squamous carcinoma cells, we assessed the radiosensitivities of the TE-5, TE-9 and TE-12 cloneA1 cell lines. TE-12 cloneA1 cells showed significantly greater susceptibility to radiotherapy at 5 and 10Gy than either TE-5 or TE-9 cells. Consistent with that finding, 24h after irradiation (5Gy), TE-12 cloneA1 cells showed higher levels of caspase 3/7 activity than TE-5 or TE-9 cells. When we used DNA microarrays to compare the gene expression profiles of TE-5 and TE-12 cloneA1 cells, we found that the mRNA and protein expression of insulin-like growth factor binding protein 3 (IGFBP3) and Bcl-2-associated athanogene 1 (BAG1) was five or more times higher in TE-12 cloneA1 cells than TE-5 cells. Conversely, knocking down expression of IGFBP3 and BAG1 mRNA in TE-12 cloneA1 cells using small interfering RNA (siRNA) significantly reduced radiosensitivity. These data suggest that IGFBP3 and BAG1 may be key markers of radiosensitivity that enhance the susceptibility of squamous cell esophageal cancer to radiotherapy. IGFBP3 and BAG1 may thus be useful targets for improved and more individualized treatments for patients with esophageal squamous cell carcinoma.
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Carcinoma de Células Escamosas/radioterapia , Proteínas de Unión al ADN/fisiología , Neoplasias Esofágicas/radioterapia , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/fisiología , Tolerancia a Radiación/genética , Factores de Transcripción/fisiología , Apoptosis/genética , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Técnicas de Silenciamiento del Gen , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Factores de Transcripción/genéticaRESUMEN
Both liver epithelial and oval cells are believed to be liver stem cells. We investigated the identification by producing monoclonal antibodies against liver epithelial cells. Monoclonal antibodies against hepatic stem-like cells (HSL cells) have been selected to follow the hepatic stem cells during hepatic regeneration and developmental changes in the liver. Monoclonal antibodies were induced by immunization of BALB/c mice with HSL cells established from the epithelial cells of the adult rat liver. The hybridomas were screened by indirect immunofluorescence staining of HSL cells. We produced a unique monoclonal antibody against HSL cells, MabH, which specifically recognizes liver epithelial cells. MabH did not react with liver parenchymal cells but did react with bile ductule cells under normal conditions in the adult liver. This antibody also reacted with oval cell lines and with the oval cells that appeared during liver regeneration. In addition, fetal liver cells showed immunoreactivity with MabH. Although the level of staining decreased after birth, some cells in the portal area remained highly reactive. These results suggested that liver epithelial cells, oval cells, and fetal liver cells possess a common cell marker of liver stem cells.
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Anticuerpos Monoclonales , Hibridomas/inmunología , Hígado , Células Madre/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Conductos Biliares/citología , Conductos Biliares/inmunología , Línea Celular , Células Epiteliales/inmunología , Femenino , Hígado/citología , Hígado/inmunología , Regeneración Hepática , Ratones , Ratones Endogámicos BALB C , Ratas , Ratas Sprague-DawleyRESUMEN
OBJECTIVE: Eosinophils are tissue-dwelling immune cells. Accumulating evidence indicates that a type of cell death termed ETosis is an important cell fate involved in the pathophysiology of inflammatory diseases. Although the critical role of eosinophils in eosinophilic granulomatosis with polyangiitis (EGPA; formerly Churg-Strauss syndrome) is well established, the presence of eosinophil ETosis (EETosis) is poorly understood. We undertook this study to better understand the characteristics of EETosis. METHODS: In vitro studies using blood-derived eosinophils were conducted to characterize EETosis. The occurrence of EETosis in tissues from patients with EGPA was studied by immunostaining and electron microscopy. Serum concentrations of eosinophil-derived proteins in healthy controls, patients with asthma, and EGPA patients with active disease or with disease in remission (n = 15 per group) were examined. RESULTS: EETosis was reliant on reactive oxygen species and peptidylarginine deiminase type 4-dependent histone citrullination, resulting in the cytolytic release of net-like eosinophil extracellular traps, free galectin-10, and membrane-bound intact granules. The signature of EETosis, including loss of cytoplasmic galectin-10 and deposition of granules, was observed in eosinophils infiltrating various tissues from EGPA patients. Serum eosinophil granule proteins and galectin-10 levels were increased in EGPA and positively correlated with disease activity as assessed by the Birmingham Vasculitis Activity Score (r = 0.8531, P < 0.0001 for galectin-10). When normalized to blood eosinophil counts, this correlation remained for galectin-10 (r = 0.7168, P < 0.0001) but not for granule proteins. Galectin-10 levels in active EGPA positively correlated with serum interleukin-5 levels. CONCLUSION: Eosinophils infiltrating diseased tissues in EGPA undergo EETosis. Considering the exclusive expression and large pool of cytoplasmic galectin-10 in eosinophils, elevated serum galectin-10 levels in patients with EGPA might reflect the systemic occurrence of cytolytic EETosis.
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Muerte Celular/fisiología , Eosinófilos/metabolismo , Galectinas/sangre , Granulomatosis con Poliangitis/metabolismo , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Idiopathic pure red cell aplasia (PRCA) and secondary PRCA associated with thymoma and large granular lymphocyte leukemia are generally considered to be immune-mediated. The PRCA2004/2006 study showed that poor responses to immunosuppression and anemia relapse were associated with death. PRCA may represent the prodrome to MDS. Thus, clonal hematopoiesis may be responsible for treatment failure. We investigated gene mutations in myeloid neoplasm-associated genes in acquired PRCA. We identified 21 mutations affecting amino acid sequences in 11 of the 38 adult PRCA patients (28.9%) using stringent filtering of the error-prone sequences and SNPs. Four PRCA patients showed 7 driver mutations in TET2, DNMT3A and KDM6A, and 2 PRCA patients carried multiple mutations in TET2. Five PRCA patients had mutations with high VAFs exceeding 0.3. These results suggest that clonal hematopoiesis by stem/progenitor cells might be related to the pathophysiology of chronic PRCA in certain adult patients.
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Hematopoyesis Clonal , Aplasia Pura de Células Rojas/patología , Adulto , Anciano , Anciano de 80 o más Años , Anemia Aplásica/genética , Línea Celular , Humanos , Leucemia Mieloide/genética , Persona de Mediana Edad , Mutación/genética , Aplasia Pura de Células Rojas/genéticaRESUMEN
Regenerating gene (REG) I plays important roles in cancer cell biology. The purpose of this study was to determine whether REG I affects cytokine production in cancer cells. We transfected TE-5 and TE-9 squamous esophageal cancer cells with REG Ialpha and Ibeta and examined its effects on cytokine expression. We found that transfecting TE-5 and TE-9 cells with REG I Ialpha and Ibeta led to significantly increased expression of interleukin (IL)-6 mRNA and protein, but it had little or no effect on expression of IL-2, IL-4, IL-5, IL-10, IL-12, IL-13, IL-17A, interferon-gamma, tumor necrosis factor-alpha, granulocyte-colony stimulating factor or transforming growth factor-beta1. The elevated IL-6 expression seen in REG Ialpha transfectants was silenced by small interfering RNA-mediated knockdown. These finding suggest that REG I may act through IL-6 to exert effects on squamous esophageal cancer cell biology.
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Neoplasias Esofágicas/metabolismo , Interleucina-6/biosíntesis , Litostatina/metabolismo , Neoplasias de Células Escamosas/metabolismo , Línea Celular Tumoral , Humanos , Litostatina/genética , TransfecciónRESUMEN
Wilson disease is a genetic disorder characterized by the accumulation of copper in the body by defective biliary copper excretion. Wilson disease gene product (ATP7B) functions in copper incorporation to ceruloplasmin (Cp) and biliary copper excretion. However, copper metabolism in hepatocytes has been still unclear. Niemann-Pick disease type C (NPC) is a lipid storage disorder and the most commonly mutated gene is NPC1 and its gene product NPC1 is a late endosome protein and regulates intracellular vesicle traffic. In the present study, we induced NPC phenotype and examined the localization of ATP7B and secretion of holo-Cp, a copper-binding mature form of Cp. The vesicle traffic was modulated using U18666A, which induces NPC phenotype, and knock down of NPC1 by RNA interference. ATP7B colocalized with the late endosome markers, but not with the trans-Golgi network markers. U18666A and NPC1 knock down decreased holo-Cp secretion to culture medium, but did not affect the secretion of other secretory proteins. Copper accumulated in the cells after the treatment with U18666A. These findings suggest that ATP7B localizes in the late endosomes and that copper in the late endosomes is transported to the secretory compartment via NPC1-dependent pathway and incorporated into apo-Cp to form holo-Cp.
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Adenosina Trifosfatasas/metabolismo , Proteínas Portadoras/fisiología , Proteínas de Transporte de Catión/metabolismo , Cobre/metabolismo , Endosomas/metabolismo , Glicoproteínas de Membrana/fisiología , Subunidades gamma de Complejo de Proteína Adaptadora/metabolismo , Adenosina Trifosfatasas/genética , Androstenos/farmacología , Anticolesterolemiantes/farmacología , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Proteínas Portadoras/genética , Proteínas de Transporte de Catión/genética , Línea Celular Tumoral , Ceruloplasmina/metabolismo , ATPasas Transportadoras de Cobre , Humanos , Péptidos y Proteínas de Señalización Intracelular , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Proteína 2 de la Membrana Asociada a los Lisosomas , Proteínas de Membrana de los Lisosomas/metabolismo , Glicoproteínas de Membrana/genética , Mutación , Proteína Niemann-Pick C1 , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión a GTP rab7RESUMEN
Angiotensin-converting enzyme 2 (ACE2) is critically involved in cardiovascular physiology and pathology, and is currently clinically evaluated to treat acute lung failure. Here we show that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp. B38, is an ACE2-like enzyme to decrease angiotensin II levels in mice. In protein 3D structure analysis, B38-CAP homolog shares structural similarity to mammalian ACE2 with low sequence identity. In vitro, recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7, as well as other known ACE2 target peptides. Treatment with B38-CAP suppressed angiotensin II-induced hypertension, cardiac hypertrophy, and fibrosis in mice. Moreover, B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Our data identify the bacterial B38-CAP as an ACE2-like carboxypeptidase, indicating that evolution has shaped a bacterial carboxypeptidase to a human ACE2-like enzyme. Bacterial engineering could be utilized to design improved protein drugs for hypertension and heart failure.
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Carboxipeptidasas/farmacología , Cardiomegalia/tratamiento farmacológico , Fibrosis/tratamiento farmacológico , Hipertensión/tratamiento farmacológico , Paenibacillus/enzimología , Peptidil-Dipeptidasa A/genética , Angiotensina II/metabolismo , Enzima Convertidora de Angiotensina 2 , Animales , Cardiomegalia/patología , Modelos Animales de Enfermedad , Fibrosis/patología , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/prevención & control , Hipertensión/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Peptidil-Dipeptidasa A/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Contemporary strategies in tooth repair markedly rely on the newest findings on the cellular and biological components of dental development. Among several identified bioactive molecules, neurotrophins were recently proposed to affect tooth germ cell proliferation, differentiation, and cell-extracellular matrix interactions. The present study attempted to explore the effect of nerve growth factor (NGF) on a spontaneously immortalized dental papilla mesenchymal cell line. NGF induced differentiation of odontoblast-lineage cells with subsequent biomineralization in vitro. Here we showed that normalized transcript levels of tissue-specific markers such as DSPP and DMP1 were elevated significantly, indicating cell differentiation and maturation processes. We performed innovative gene expression analysis of TM14, a matricellular protein and novel member of the fibulin family. TM14 expression followed a pattern similar to that of DMP1, which suggests its important role in cell-matrix and intercellular interactions during dentin calcification. Alkaline phosphatase enzyme assay confirmed the extracellular matrix calcifications in NGF-supplemented groups. Thus, NGF was characterized as a potent promoter of mineralization during dentin formation. For the first time, we included TM14 in odontoblast genotype analysis and proved that NGF also promotes in vitro odontoblast differentiation. Collectively, these results highlight the importance of NGF during tooth morphogenesis, as well as urge the elaboration of complex epithelial-mesenchymal tissue cultures, where further elucidation of the signaling factor network could be completed.
Asunto(s)
Diferenciación Celular , Factor de Crecimiento Nervioso/fisiología , Odontoblastos/citología , Animales , Calcificación Fisiológica , Proteínas de Unión al Calcio/análisis , Dentina/crecimiento & desarrollo , Proteínas de la Matriz Extracelular/análisis , Ratones , Ratones Endogámicos C57BL , Morfogénesis , Odontogénesis , Fosfoproteínas/análisis , Diente/crecimiento & desarrolloRESUMEN
Recent progress in cancer biology research has shown that abnormal proliferation in tumor cells can be attributed to aberrations in cell cycle regulation, especially in G1 phase. During the course of searching for microbial metabolites that affect cell cycle distribution, we have found that simaomicin α, a polycyclic xanthone antibiotic, arrests the cell cycle at G1 phase. Treatment of T-cell leukemia Jurkat cells with 3 nM simaomicin α induced an increase in the number of cells in G1 and a decrease in those in G2M phase. Cell cycle aberrations induced by simaomicin α were also detected in colon adenocarcinoma HCT15 cells. Simaomicin α had antiproliferative activities in various tumor cell lines with 50% inhibitory concentration values in the range of 0.319 nM. Furthermore, simaomicin α induced an increase in cellular caspase-3 activity and DNA fragmentation, indicating that simaomicin α promotes apoptosis. The retinoblastoma protein phosphorylation status of simaomicin α-treated cell lysate was lower than that of control cells, suggesting that the target molecule of simaomicin α is in a pathway upstream of retinoblastoma protein phosphorylation. In the course of evaluating polycyclic xanthone antibiotics structurally related to simaomicin α, we also found that cervinomycin A1 stimulated accumulation of treated cells in G1 phase. These results indicate that the polycyclic xanthones, including simaomicin α and cervinomycin A1, may be candidate cancer chemotherapeutic agents.
Asunto(s)
Fase G1/efectos de los fármacos , Isoquinolinas/farmacología , Neoplasias/tratamiento farmacológico , Proteína de Retinoblastoma/metabolismo , Apoptosis/efectos de los fármacos , Western Blotting , Proliferación Celular/efectos de los fármacos , Humanos , Neoplasias/patología , Fosforilación/efectos de los fármacos , Células Tumorales CultivadasRESUMEN
Regenerating gene (Reg) I has been identified as a regenerative/proliferative factor for pancreatic islet cells. We examined Reg I expression in the regenerating liver of a rat model that had been administered 2-acetylaminofluorene and treated with 70% partial hepatectomy (2-AAF/PH model), where hepatocyte and cholangiocyte proliferation was suppressed and the hepatic stem cells and/or hepatic progenitor cells were activated. In a detailed time course study of activation of hepatic stem cells in the 2-AAF/PH model, utilizing immunofluorescence staining with antibodies of Reg I and other cell-type-specific markers, we found that Reg I-expressing cells are present in the bile ductules and increased during regeneration. Reg I-expressing cells were colocalized with CK19, OV6, and AFP. These results demonstrate that Reg I is significantly upregulated in the liver of the 2-AAF/PH rat model, accompanied by the formation of bile ductules during liver regeneration.
Asunto(s)
Litostatina/metabolismo , Regeneración Hepática , Hígado/metabolismo , Animales , Conductos Biliares/metabolismo , Expresión Génica , Litostatina/biosíntesis , Litostatina/genética , Masculino , Modelos Animales , Ratas , Ratas Endogámicas F344 , Regulación hacia ArribaRESUMEN
Identification of reliable markers of chemo- and radiosensitivity and the key molecules that enhance the susceptibility of squamous esophageal cancer cells to anticancer treatments would be highly desirable. To test whether regenerating gene (REG) I expression enhances chemo- and radiosensitivity in esophageal squamous cell carcinoma cells, we used MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assays to compare the chemo- and radiosensitivities of untransfected TE-5 and TE-9 cells with those of cells stably transfected with REG Ialpha and Ibeta. We then used flow cytometry to determine whether REG I expression alters cell cycle progression. No REG I mRNA or protein were detected in untransfected TE-5 and TE-9 cells. Transfection with REG Ialpha and Ibeta led to strong expression of both REG I mRNA and protein in TE-5 and TE-9 cells, which in turn led to significant increases in both chemo- and radiosensitivity. Cell cycle progression was unaffected by REG I expression. REG I thus appears to enhance the chemo- and radiosensitivity of squamous esophageal cancer cells, which suggests that it may be a useful target for improved and more individualized treatments for patients with esophageal squamous cell carcinoma.