2. Academic Validation
  3. Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System

Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System

  • Cells. 2022 May 18;11(10):1677. doi: 10.3390/cells11101677.
Ryosuke Negoro 1,  Mitsuki Tasaka 2,  Sayaka Deguchi 3,  Kazuo Takayama 3 4,  Takuya Fujita 1 2 5
Affiliations

Affiliations

  • 1 Laboratory of Molecular Pharmacokinetics, College of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu 525-8577, Japan.
  • 2 Laboratory of Molecular Pharmacokinetics, Graduate School of Pharmaceutical Sciences, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu 525-8577, Japan.
  • 3 Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan.
  • 4 Japan Agency for Medical Research and Development-Core Research for Evolutionary Medical Science and Technology (AMED-CREST), Japan Agency for Medical Research and Development, 1-7-1 Otemachi, Chiyoda-ku, Tokyo 100-0004, Japan.
  • 5 Research Center for Drug Discovery and Development, Ritsumeikan University, 1-1-1 Noji-Higashi, Kusatsu 525-8577, Japan.
PMID: 35626714 DOI: 10.3390/cells11101677
Abstract

HepG2 cells are an inexpensive hepatocyte model that can be used for repeated experiments, but HepG2 cells do not express major cytochrome P450s (CYPs) and UDP glucuronosyltransferase family 1 member A1 (UGT1A1). In this study, we established CYP3A4-POR-UGT1A1-CYP1A2-CYP2C19-CYP2C9-CYP2D6 (CYPs-UGT1A1) knock-in (KI)-HepG2 cells using a PITCh system to evaluate whether they could be a new hepatocyte model for pharmaceutical studies. To evaluate whether CYPs-UGT1A1 KI-HepG2 cells express and function with CYPs and UGT1A1, gene expression levels of CYPs and UGT1A1 were analyzed by using Real-Time PCR, and metabolites of CYPs or UGT1A1 substrates were quantified by HPLC. The expression levels of CYPs and UGT1A1 in the CYPs-UGT1A1 KI-HepG2 cells were comparable to those in primary human hepatocytes (PHHs) cultured for 48 h. The CYPs and UGT1A1 activity levels in the CYPs-UGT1A1 KI-HepG2 cells were much higher than those in the wild-type (WT)-HepG2 cells. These results suggest that the CYPs-UGT1A1 KI-HepG2 cells expressed functional CYPs and UGT1A1. We also confirmed that the CYPs-UGT1A1 KI-HepG2 cells were more sensitive to drug-induced liver toxicity than the WT-HepG2 cells. CYPs-UGT1A1 KI-HepG2 cells could be used to predict drug metabolism and drug-induced liver toxicity, and they promise to be a helpful new hepatocyte model for drug discovery research.

Keywords

CRISPR-Cas9; CYP1A2; CYP2C19; CYP2C9; CYP2D6; CYP3A4; HepG2 cell; PITCh system; POR; UGT1A1; drug-metabolizing enzyme; genome editing; primary human hepatocytes.

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