RESUMEN
It has recently been reported that cholangiocyte organoids can be established from primary human hepatocytes. The purpose of this study was to culture the organoids in monolayers on inserts to investigate the biliary excretory capacity of drugs. Cholangiocyte organoids prepared from hepatocytes had significantly higher mRNA expression of CK19, a bile duct epithelial marker, compared to hepatocytes. The organoids also expressed mRNA for efflux transporters involved in biliary excretion of drugs, P-glycoprotein (P-gp), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP). The subcellular localization of each protein was observed. These results suggest that the membrane-cultured cholangiocyte organoids are oriented with the upper side being the apical membrane side (A side, bile duct lumen side) and the lower side being the basolateral membrane side (B side, hepatocyte side), and that each efflux transporter is localized to the apical membrane side. Transport studies showed that the permeation rate from the B side to the A side was faster than from the A side to the B side for the substrates of each efflux transporter, but this directionality disappeared in the presence of inhibitor of each transporter. In conclusion, the cholangiocyte organoid monolayer system has the potential to quantitatively evaluate the biliary excretion of drugs. The results of the present study represent an unprecedented system using human cholangiocyte organoids, which may be useful as a screening model to directly quantify the contribution of biliary excretion to the clearance of drugs.
Asunto(s)
Eliminación Hepatobiliar , Proteínas Asociadas a Resistencia a Múltiples Medicamentos , Humanos , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Hepatocitos/metabolismo , ARN Mensajero/metabolismoRESUMEN
The aim of this work is to develop a new assay system for screening biliary excretion drugs. When monolayers of human liver-derived cell lines HepG2 and Huh-7 were grown on an insert membrane, the efflux ratio (ER: ratio of the apparent permeability coefficient in the basal-to-apical direction (Papp,B-to-A) to that in the apical to basal direction (Papp,A-to-B)) of sulfobromophthalein (BSP), a model substrate of multidrug resistance-associated protein 2 (MRP2), was greater than 1.0, indicating transport of BSP in the efflux direction. The efflux transport was significantly suppressed by MK-571, an inhibitor of MRPs, in both cell lines. Expression of MRP2 mRNA in HepG2 and Huh-7 was 3.5- and 1.4-fold higher, respectively, than in primary human hepatocytes, while expression of P-glycoprotein and breast cancer resistance protein mRNAs was markedly lower, supporting the idea that MRP2 is the main mediator of directional BSP transport in this assay system. The advantage of our system is the potential to quantitatively evaluate biliary excretion of MRP2 substrates in vitro.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP , Proteínas de Neoplasias , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Transporte Biológico , Línea Celular , Humanos , Hígado/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas de Neoplasias/metabolismoRESUMEN
The NanoCulture Plate (NCP) is a novel microstructural plate designed as a base for the three-dimensional culture of cells/tissues. This study examined whether or not the metabolic capability of human primary hepatocytes is well maintained during culture on NCPs. The hepatocytes formed aggregates after seeding and their ATP content was well maintained during culture for 21 days. Expression of CYP1A2, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 mRNAs was detected throughout the 21-day culture period. Addition of CYP substrate drugs (midazolam, diclofenac, lamotrigine and acetaminophen) resulted in the formation of multiple metabolites with a corresponding decrease in the amounts of the unchanged compounds. The inducers omeprazole, phenobarbital and rifampicin increased the levels of CYP1A2, 2B6 and 3A4 mRNAs by 110-fold, 12.5-fold and 5.4-fold, respectively, at day 2, compared with control human hepatocytes. CYP activities were also increased at 2 days after inducer treatment (CYP1A2, 2.2-fold; CYP2B6, 20.6-fold; CYP3A4, 3.3-fold). The results indicate that the hepatocyte spheroids on NCP have detectable and inducible metabolic abilities during the 7-day culture period.
Asunto(s)
Hepatocitos/metabolismo , Acetaminofén/farmacología , Adenosina Trifosfato/metabolismo , Arilsulfotransferasa/genética , Técnicas de Cultivo de Célula , Células Cultivadas , Inductores de las Enzimas del Citocromo P-450/farmacología , Sistema Enzimático del Citocromo P-450/genética , Diclofenaco/farmacología , Glucuronosiltransferasa/genética , Humanos , Lamotrigina , Midazolam/farmacología , Omeprazol/farmacología , ARN Mensajero/metabolismo , Rifampin/farmacología , Triazinas/farmacologíaRESUMEN
Epithelial-mesenchymal transition (EMT) is a crucial pathological event in cancer, particularly in tumor cell budding and metastasis. Therefore, control of EMT can represent a novel therapeutic strategy in cancer. Here, we introduce an innovative three-dimensional (3D) high-throughput screening (HTS) system that leads to an identification of EMT inhibitors. For the establishment of the novel 3D-HTS system, we chose NanoCulture Plates (NCP) that provided a gel-free micro-patterned scaffold for cells and were independent of other spheroid formation systems using soft-agar. In the NCP-based 3D cell culture system, A549 lung cancer cells migrated, gathered, and then formed multiple spheroids within 7 days. Live cell imaging experiments showed that an established EMT-inducer TGF-ß promoted peripheral cells around the core of spheroids to acquire mesenchymal spindle shapes, loss of intercellular adhesion, and migration from the spheroids. Along with such morphological change, EMT-related gene expression signatures were altered, particularly alteration of mRNA levels of ECAD/CDH1, NCAD/CDH2, VIM and ZEB1/TCF8. These EMT-related phenotypic changes were blocked by SB431542, a TGF-ßreceptor I (TGFßR1) inhibitor. Inside of the spheroids were highly hypoxic; in contrast, spheroid-derived peripheral migrating cells were normoxic, revealed by visualization and quantification using Hypoxia Probe. Thus, TGF-ß-triggered EMT caused spheroid hypoplasia and loss of hypoxia. Spheroid EMT inhibitory (SEMTIN) activity of SB431542 was calculated from fluorescence intensities of the Hypoxia Probe, and then was utilized in a drug screening of EMT-inhibitory small molecule compounds. In a pilot screening, 9 of 1,330 compounds were above the thresholds of the SEMTIN activity and cell viability. Finally, two compounds SB-525334 and SU9516 showed SEMTIN activities in a dose dependent manner. SB-525334 was a known TGFßR1 inhibitor. SU9516 was a cyclin-dependent kinase 2 (CDK2) inhibitor, which we showed also had an EMT-inhibitory activity. The half maximal inhibitory concentration (IC50) of SB-525334 and SU9516 were 0.31 µM and 1.21 µM, respectively, while IC50 of SB431542 was 2.38 µM. Taken together, it was shown that this 3D NCP-based HTS system was useful for screening of EMT-regulatory drugs.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Ensayos de Selección de Medicamentos Antitumorales/métodos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Imidazoles/farmacología , Indoles/farmacología , Benzamidas/farmacología , Hipoxia de la Célula/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dioxoles/farmacología , Ensayos de Selección de Medicamentos Antitumorales/instrumentación , Humanos , Proyectos Piloto , Esferoides Celulares , Factor de Crecimiento Transformador beta2/farmacologíaRESUMEN
Severe advanced head and neck carcinoma which can not be removed via surgical procedure combined with a large lymph node metastasis has a poor prognosis. We administered concurrent chemoradiotherapy with S-1 for a lower gingival carcinoma. As a direct result, we discovered that the treatment greatly reduced the size of tumor, and we consider that this treatment prolonged the patient.s life. The treatment results suggest that the so-called dormancy state of the tumor was continued. In this case study, radiotherapy with S-1 showed a highly effective response from the viewpoint of QOL improvement.