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

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–UGT1...

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Autores principales: Negoro, Ryosuke, Tasaka, Mitsuki, Deguchi, Sayaka, Takayama, Kazuo, Fujita, Takuya
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9140068/
https://www.ncbi.nlm.nih.gov/pubmed/35626714
http://dx.doi.org/10.3390/cells11101677
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author Negoro, Ryosuke
Tasaka, Mitsuki
Deguchi, Sayaka
Takayama, Kazuo
Fujita, Takuya
author_facet Negoro, Ryosuke
Tasaka, Mitsuki
Deguchi, Sayaka
Takayama, Kazuo
Fujita, Takuya
author_sort Negoro, Ryosuke
collection PubMed
description 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.
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spelling pubmed-91400682022-05-28 Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System Negoro, Ryosuke Tasaka, Mitsuki Deguchi, Sayaka Takayama, Kazuo Fujita, Takuya Cells Article 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. MDPI 2022-05-18 /pmc/articles/PMC9140068/ /pubmed/35626714 http://dx.doi.org/10.3390/cells11101677 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Negoro, Ryosuke
Tasaka, Mitsuki
Deguchi, Sayaka
Takayama, Kazuo
Fujita, Takuya
Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System
title Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System
title_full Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System
title_fullStr Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System
title_full_unstemmed Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System
title_short Generation of HepG2 Cells with High Expression of Multiple Drug-Metabolizing Enzymes for Drug Discovery Research Using a PITCh System
title_sort generation of hepg2 cells with high expression of multiple drug-metabolizing enzymes for drug discovery research using a pitch system
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9140068/
https://www.ncbi.nlm.nih.gov/pubmed/35626714
http://dx.doi.org/10.3390/cells11101677
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