Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: Ultrafine structure of functionally enhanced hepatocarcinoma cell lines

Kawada, Masayuki; Nagamori, Ippei; Aizaki, Hideki; Fukaya, Keisuke; Niiya, Minoru; Matsuura, Tomokazu; Sujino, H; Hasumura, Satoshi; Yashida, Hitoshi; Mizutani, S; Ikenaga, Hiroshi

doi:10.1007/s11626-998-0092-z

Cited by 64 publications

(45 citation statements)

References 13 publications

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“…Use of an RFB (RA-15, ABLE Co., Ltd., Tokyo, Japan) and mass flow controller (RAD925, ABLE Co., Ltd.) has previously been reported for the high-density, 3-dimensional mass production of cells, which attach to a matrix. 12,13 The matrix consisted of hydroxy apatite beads (diameter 1-2 mm, pore size Ͻ200 m, Asahi Optical Co., Ltd., Tokyo, Japan), with a high pore density, which gave a wide surface attachment area.…”

Section: Methodsmentioning

confidence: 99%

CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver

Iwahori

2003

Hepatology

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CYP3A is responsible for approximately 50% of the therapeutic drug-metabolizing activity in the liver. The present study was undertaken to establish the CYP3A4 inducible model for analysis of human drug metabolism using a bioartificial liver composed of the functional hepatocellular carcinoma cell (HCC) line FLC-5. A radial-flow bioreactor (RFB), which is a carrier-filled type bioreactor, was used for 3-dimensional perfusion culture of FLC-5 cells. The CYP3A4 messenger RNA (mRNA) expression level 48 hours after rifampicin treatment in the RBF was approximately 100 times higher than that in a monolayer culture. Western blot analysis also demonstrated an increase in expression of the CYP3A protein. When testosterone, a substrate for CYP3A4, was added to the rifampicin-treated cell culture, 6␤-hydroxy testosterone as a metabolite was formed. D rug-drug interactions can be categorized as mediated by metabolic inhibition and enzyme induction. Numerous studies have been conducted on metabolic inhibition. It is now possible, to some extent, to predict drug-drug interactions in vivo based on the results in vitro. Enzyme induction has been studied using experimental animals, but, because of the existence of interspecies differences, it is often difficult to extrapolate the results of animal studies directly to humans. 1 To resolve these problems, enzyme-induction experiments have been performed using human primary cell culture systems, and experimental models established using these cell cultures have been recognized to be useful in the evaluation of enzyme induction. 2 However, longterm primary culture of human hepatocytes can reduce the functions of liver enzymes, as reflected, for example, by a decrease in the level of cytochrome P450 (CYP), an enzyme mainly catalyzing phase-I reactions of drug metabolism, 4 days after isolation, making it difficult to use these cells for prolonged periods of time. 3 Furthermore, because of interracial or sex-related differences and differences in storage periods (cell viability), the drug-metabolizing activity varies greatly among cells, causing problems in reproducibility. 4,5 For these reasons, much has been expected of liver cell lines that have uniform and stable properties, can be used for long-term experiments, are highly differentiated, and retain human liver functions.

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Section: Methodsmentioning

confidence: 99%

CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver

Iwahori

2003

Hepatology

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“…HepG2, Ym17, and Yh18 cells were maintained as described previously (Swales et al, 2005). FLC7 cells were kindly provided by Dr. Nagamori (Kyorin University, Tokyo, Japan) (Kawada et al, 1998). FLC7 and Huh7 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% 200 mM glutamine, and antibiotics.…”

Section: Introductionmentioning

confidence: 99%

p38 Mitogen–Activated Protein Kinase Regulates Nuclear Receptor CAR that Activates the CYP2B6 Gene

2013

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The constitutive active/androstane receptor (CAR) regulates hepatic drug metabolism by activating genes, such as cytochrome P450, and certain transferases. p38 Mitogen-activated protein kinase (MAPK) is highly activated in human primary hepatocytes but barely in human hepatoma cell lines including HepG2 cells. Liganded-CAR induced CYP2B6 mRNA in human primary hepatocytes far more effectively than in HepG2 cells ectopically expressing CAR. In the present study, we found that activation of p38 MAPK by anisomycin potentiated induction of CYP2B6 mRNA by CAR ligand in HepG2 cells to levels observed in ligandtreated human primary hepatocytes. siRNA knockdown of p38 MAPK abrogated the ability of anisomycin to synergistically induce CYP2B6 mRNA. In addition to CYP2B6, anisomycin cotreatment potentiated an increase in CYP2A7 and CYP2C9 mRNAs but not CYP3A4 or UDP-glucuronosyltransferase 1A1 mRNAs. Thus, activated p38 MAPK is required for liganded-CAR to selectively activate a set of genes that encode drug-metabolizing enzymes. Our present results suggest that CAR-mediated induction of these enzymes cannot be understood by ligand binding alone because the specificity and magnitude of induction are codetermined by a given cell signaling, such as p38 MAPK; both physiologic and pathophysiological states of cell signaling may have a strong impact in hepatic drug-metabolizing capability during treatments.

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“…FLC7 cells (Kawada et al, 1998), a human hepatocellular carcinoma cell line, were kindly provided by Dr. S. Nagamori (Kyorin University, Tokyo, Japan). FLC7 cells were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 10% fetal bovine serum.…”

Section: Methodsmentioning

confidence: 99%

Key Structural Features of Ligands for Activation of Human Pregnane X Receptor

Kobayashi

Yamagami²,

Higuchi³

et al. 2004

Drug Metab Dispos

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ABSTRACT:The ligand-binding domain of human pregnane X receptor (hPXR) is highly hydrophobic and flexible, allowing promiscuity in accepting structurally diverse ligands. However, little information is available regarding the critical substituents of compounds involved in the activation of hPXR. The aim of this study was to determine the structure-activity relationships for hPXR-mediated transactivation by barbiturates, hydantoins, and macrolide antibiotics. Most of the barbiturates studied (mephobarbital, pentobarbital, phenobarbital, etc.) activated hPXR. However, barbital, which has a low hydrophobic moiety at the 5-position, and primidone, which has no carbonyl moiety at the 2-position, did not activate hPXR. Therefore, a hydrophobic moiety at the 5-position and a hydrogen-bond acceptor being sufficiently separated from the phenyl-ring are responsible for activation of hPXR by barbiturates. In the case of hydantoins, only mephenytoin and ethotoin, which have an alkylchain at the R1-position, strongly activated hPXR at 300 M. Phenytoin and 5-(4-methylphenyl)-5-phenylhydantoin, which contain a phenyl or methylphenyl group at both R2-and R3-positions, also activated hPXR, whereas 5-(4-hydroxyphenyl)-5-phenylhydantoin did not activate the receptor. These results suggest that the presence of an alkyl-chain at the R1-position and the presence of bulky and hydrophobic moieties at both R2-and R3-positions are important factors for activation of hPXR by hydantoins. In the case of macrolide antibiotics, troleandomycin, but not oleandomycin, showed significant activation of hPXR. Therefore, triacetate esterification of oleandomycin might increase the hydrophobicity and enhance the activation of hPXR. These findings suggest that hydrophobicity of the ligand and adequate distance between the hydrogen-bond acceptor and the hydrophobic group are important for hPXR activation.

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Massive culture of human liver cancer cells in a newly developed radial flow bioreactor system: Ultrafine structure of functionally enhanced hepatocarcinoma cell lines

Cited by 64 publications

References 13 publications

CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver

CYP3A4 inducible model for in vitro analysis of human drug metabolism using a bioartificial liver

p38 Mitogen–Activated Protein Kinase Regulates Nuclear Receptor CAR that Activates the CYP2B6 Gene

Key Structural Features of Ligands for Activation of Human Pregnane X Receptor

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