P289 Induced Expressions and Activities of Drug-Metabolizing Enzymes in the Human Hepatocellular Carcinoma Cell Line Cultured On the Micro-Space Cell Culture Plates

Akane Yoshida , Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
Kaoru Kobayashi , Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
Yoko Ejiri , Tsukuba Research Center, Kuraray Co., Ltd., Tsukuba, Japan
Sachiko Takagi , Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
Hanaka Mimura , Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
Masaya Hosoda , Tsukuba Research Center, Kuraray Co., Ltd., Tsukuba, Japan
Kan Chiba , Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan
Human hepatocellular carcinoma cell lines cultured in monolayer have been limited to use for drug metabolism study because of negligible activities of drug-metabolizing enzymes such as cytochrome P450s (CYPs). Several studies showed that the expression levels of CYPs in human hepatocellular carcinoma cells were increased by three-dimensional culture. However, the three-dimensional culture systems are generally hard to handle. To reduce technical complexity 24-well plates arrayed with uniform micro-sized compartments on the bottom (micro-space cell culture plates) have been developed as a novel system. The purpose of the present study was to evaluate whether human hepatocellular carcinoma FLC4 cells cultured on the micro-space cell culture plates are useful for drug metabolism study. In this study, we compared expression levels and activities of drug-metabolizing enzymes in FLC4 cells cultured on the micro-space cell culture plates with those in monolayer FLC4 cells. When FLC4 cells were cultured on the micro-space cell culture plates, the cells formed spheroids. Results of DNA microarray analyses showed that mRNA expression levels of various drug-metabolizing enzymes including CYPs, UDP-glucuronosyltransferases (UGTs), sulfotransferases and glutathione S-transferases in spheroid cells were higher than those in monolayer cells. Since these genes were expressed in human liver tissues at the high levels, it was thought that the expression profiles of drug-metabolizing enzymes in spheroid cells were similar to those in human liver tissues. Results of real-time PCR analyses showed that expression levels of CYP1A2, CYP2C9, CYP3A4 and UGT1A1 mRNAs in the spheroid cells were remarkably higher than those in monolayer cells. When FLC4 cells were treated with diclofenac (CYP2C9 substrate) or triazolam (CYP3A substrate), significant formations of their metabolites were detected in the medium of spheroid cells. On the other hand, the formations of metabolites were negligible in the medium of monolayer cells. In addition, expression levels of pregnane X receptor (PXR) and constitutive androstane receptor (CAR) mRNAs in spheroid cells were higher than those in monolayer cells. In only spheroid cells, expression levels of CYP3A4 and CYP2B6 mRNAs were also induced by rifampicin (PXR ligand) and CITCO (CAR ligand), respectively. These results suggest that the micro-space cell culture plates are useful tools for drug metabolism study using the human hepatocellular carcinoma cell line.
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