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Anticancer Res ; 40(1): 169-176, 2020 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-31892565


BACKGROUND/AIM: Cancer stem cells (CSCs) are considered to be one of the causes of tumor recurrence after chemotherapy. The purpose of our study was to isolate CSCs from human colorectal cancer cell (CRC) lines. MATERIALS AND METHODS: Nine CRC lines were screened based on the expression level of potential CSC markers to identify putative CSCs. Tumor formation capacity in immunodeficient mice was compared with that of their counterparts. Stemness, differentiation potency and sensitivity to 5-fluorouracil (5-FU), in vitro, were also assessed. Microarray analysis was used to characterize the features of the putative CSCs. RESULTS: COLO 201 cells were separated into two populations based on CD44 expression. CD44 positive (CD44+) cells showed significantly higher tumor formation capacity than CD44- cells in immunodeficient mice. CD44+ cells also possessed stemness properties and lower sensitivity to 5-FU in vitro. Moreover, cancer stemness and chemoresistance-related genes were highly up-regulated in CD44+ cells. CONCLUSION: CD44+ COLO 201 cells possessed the features of CSCs; therefore, the present CSC model could serve as a valuable tool to accelerate CSC research.

Receptores de Hialuranos/metabolismo , Células Madre Neoplásicas/metabolismo , Animales , Biomarcadores , Biomarcadores de Tumor , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Modelos Animales de Enfermedad , Citometría de Flujo , Fluorouracilo/farmacología , Xenoinjertos , Humanos , Receptores de Hialuranos/genética , Ratones
Exp Hematol ; 59: 30-39.e2, 2018 03.
Artículo en Inglés | MEDLINE | ID: mdl-29274361


Lusutrombopag (S-888711), an oral small-molecule thrombopoietin receptor (TPOR) agonist, has gained first approval as a drug to treat thrombocytopenia of chronic liver disease in patients undergoing elective invasive procedures in Japan. Preclinical studies were performed to evaluate its efficacy against megakaryopoiesis and thrombopoiesis. To investigate the proliferative activity and efficacy of megakaryocytic colony formation via human TPOR, lusutrombopag was applied to cultured human c-Mpl-expressing Ba/F3 (Ba/F3-hMpl) cells and human bone marrow-derived CD34-positive cells, respectively. Lusutrombopag caused a robust increase in Ba/F3-hMpl cells by activating pathways in a manner similar to that of thrombopoietin and induced colony-forming units-megakaryocyte and polyploid megakaryocytes in human CD34-positive cells. Because lusutrombopag has high species specificity for human TPOR, there was no suitable experimental animal model for drug evaluation, except for immunodeficient mouse-based xenograft models. Therefore, a novel genetically modified knock-in mouse, TPOR-Ki/Shi, was developed by replacing mouse Mpl with human-mouse chimera Mpl. In TPOR-Ki/Shi mice, lusutrombopag significantly increased circulating platelets in a dose-dependent manner during 21-day repeated oral administration. Histopathological study of the TPOR-Ki/Shi mice on day 22 also revealed a significant increase in megakaryocytes in the bone marrow. These results indicate that lusutrombopag acts on human TPOR to upregulate differentiation and proliferation of megakaryocytic cells, leading to platelet production.

Proliferación Celular/efectos de los fármacos , Cinamatos/farmacología , Megacariocitos/metabolismo , Modelos Biológicos , Receptores de Trombopoyetina/agonistas , Tiazoles/farmacología , Animales , Plaquetas/citología , Plaquetas/metabolismo , Línea Celular , Evaluación Preclínica de Medicamentos , Técnicas de Sustitución del Gen , Humanos , Megacariocitos/citología , Ratones , Ratones Transgénicos , Receptores de Trombopoyetina/genética , Receptores de Trombopoyetina/metabolismo
Haematologica ; 93(10): 1495-504, 2008 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-18728031


BACKGROUND: Thrombocytopenia is a common problem in the management of patients with cancer and other conditions that affect hematopoietic cells. In previous clinical trials, the polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor increased platelet counts in patients with idiopathic thrombocytopenic purpura and solid tumors undergoing chemotherapy. However, antibodies to polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor develop in healthy volunteers and patients undergoing chemotherapy and cross-react with endogenous thrombopoietin. As a result, clinical development of polyethylene-glycol-conjugated recombinant human megakaryocyte growth and development factor was discontinued in 1998. The aim of this study was to identify an orally bioavailable human Mpl activator that does not develop autoantibodies against endogenous thrombopoietin. DESIGN AND METHODS: We screened our chemical library and created a novel non-peptidyl thrombopoietin receptor, Mpl activator named butyzamide. We evaluated the effect of butyzamide on megakaryopoiesis in vitro using Ba/F3 cells expressing Mpl and human hematopoietic stem cells. For the evaluation of its in vivo effect, we administered butyzamide orally to immunodeficient NOD/Shi-scid,IL-2R gamma(null) (NOG) mice transplanted with human fetal liver-derived CD34(+) cells and investigated the production of human platelets. RESULTS: Butyzamide specifically reacted with human Mpl and activated the same signal transduction pathway as thrombopoietin. However, unlike thrombopoietin, butyzamide did not react with murine Mpl and was shown to require the histidine residue in the transmembrane domain of Mpl for its agonistic activity. Butyzamide induced colony-forming unit-megakaryocytes and polyploid megakaryocytes from human CD34(+) hematopoietic progenitor cells, and its effects were comparable to those of thrombopoietin. When butyzamide was administered orally at the doses of 10 and 50 mg/kg for 20 days to NOG mice transplanted with human fetal liver-derived CD34(+) cells, the human platelet count increased by 6.2- and 22.9-fold, respectively. CONCLUSIONS: Butyzamide is an orally bioavailable human Mpl activator, and appears to have potential for clinical development as a therapeutic agent for patients with thrombocytopenia.

Diferenciación Celular/efectos de los fármacos , Megacariocitos/efectos de los fármacos , Megacariocitos/metabolismo , Receptores de Trombopoyetina/metabolismo , Tiazoles/farmacología , Animales , Antígenos CD34/metabolismo , Plaquetas/citología , Plaquetas/efectos de los fármacos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Humanos , Megacariocitos/citología , Metacrilatos , Ratones , Estructura Molecular , Péptidos/farmacología , Agregación Plaquetaria/efectos de los fármacos , Receptores de Trombopoyetina/genética , Transducción de Señal/efectos de los fármacos , Tiazoles/química , Trombopoyetina/agonistas , Trombopoyetina/metabolismo
J Biol Chem ; 282(24): 17908-20, 2007 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-17442667


Mannan-binding protein (MBP) is a C-type mammalian lectin specific for mannose and N-acetylglucosamine. MBP is mainly synthesized in the liver and occurs naturally in two forms, serum MBP (S-MBP) and intracellular MBP (I-MBP). S-MBP activates complement in association with MBP-associated serine proteases via the lectin pathway. Despite our previous study (Mori, K., Kawasaki, T., and Yamashina, I. (1984) Arch. Biochem. Biophys. 232, 223-233), the subcellular localization of I-MBP and its functional implication have not been clarified yet. Here, as an extension of our previous studies, we have demonstrated that the expression of human MBP cDNA reproduces native MBP differentiation of S-MBP and I-MBP in human hepatoma cells. I-MBP shows distinct accumulation in cytoplasmic granules, and is predominantly localized in the endoplasmic reticulum (ER) and involved in COPII vesicle-mediated ER-to-Golgi transport. However, the subcellular localization of either a mutant (C236S/C244S) I-MBP, which lacks carbohydrate-binding activity, or the wild-type I-MBP in tunicamycin-treated cells shows an equally diffuse cytoplasmic distribution, suggesting that the unique accumulation of I-MBP in the ER and COPII vesicles is mediated by an N-glycan-lectin interaction. Furthermore, the binding of I-MBP with glycoprotein intermediates occurs in the ER, which is carbohydrate- and pH-dependent, and is affected by glucose-trimmed high-mannose-type oligosaccharides. These results strongly indicate that I-MBP may function as a cargo transport lectin facilitating ER-to-Golgi traffic in glycoprotein quality control.

Proteína gp120 de Envoltorio del VIH/metabolismo , Glicoproteínas de la Membrana Asociadas a los Lisosomas/metabolismo , Lectina de Unión a Manosa/metabolismo , Isoformas de Proteínas/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico/fisiología , Conformación de Carbohidratos , Secuencia de Carbohidratos , Carcinoma Hepatocelular , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteína gp120 de Envoltorio del VIH/genética , Humanos , Neoplasias Hepáticas , Lectina de Unión a Manosa/genética , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/metabolismo , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Fracciones Subcelulares/metabolismo , Resonancia por Plasmón de Superficie , Tapsigargina/metabolismo