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      Effects of Saikosaponin D on CYP1A2 and CYP2D6 in HepaRG Cells

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          Bupleurum is one of the most important traditional Chinese medicines and an ingredient in many compound preparations. It is widely used together with other drugs in clinical practice, and thus there is great potential for drug–drug interactions. Saikosaponin D (SsD) is a major bioactive triterpenoid saponin extracted from Bupleurum with anti-inflammatory, anticancer, antioxidative, and antihepatic fibrosis effects. Effects of the main components of Bupleurum on cytochromes P450 (CYPs) need to be clarified in the clinical application of combination therapies of formulations containing SsD or Bupleurum.


          This study aimed to investigate the effects of SsD on the CYP1A2 and CYP2D6 mRNAs, protein expression, and relative enzyme activities in HepaRG cells.


          HepaRG cells were cultured with SsD at concentrations of 0.5, 1, 5 and 10 μM for 72 hours. mRNA and protein expression of CYP1A2 and CYP2D6 were analyzed with real-time PCR and Western blot analysis. Relative enzyme activities were analyzed with HPLC based on consumption of the specific probe substrate.


          SsD significantly induced expression of mRNA and increased relative activity of CYP1A2 in HepaRG cells after the cells had been treated with SsD at concentrations of 1, 5 and 10 μM. SsD also induced protein expression of CYP1A2 at concentrations of 5 and 10 μM. SsD exhibited an inductive effect on CYP2D6 mRNA and protein expression, while increasing the relative activity of CYP2D6 at concentrations of 5 and 10 μM.


          This study is the first to investigate the effect of SsD on CYP1A2 and CYP2D6 in HepaRG cells, and the results may provide some useful information on potential drug–drug interactions related to clinical preparations containing SsD or Bupleurum.

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          Most cited references 47

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          Cytochrome P450 enzymes in drug metabolism: regulation of gene expression, enzyme activities, and impact of genetic variation.

          Cytochromes P450 (CYP) are a major source of variability in drug pharmacokinetics and response. Of 57 putatively functional human CYPs only about a dozen enzymes, belonging to the CYP1, 2, and 3 families, are responsible for the biotransformation of most foreign substances including 70-80% of all drugs in clinical use. The highest expressed forms in liver are CYPs 3A4, 2C9, 2C8, 2E1, and 1A2, while 2A6, 2D6, 2B6, 2C19 and 3A5 are less abundant and CYPs 2J2, 1A1, and 1B1 are mainly expressed extrahepatically. Expression of each CYP is influenced by a unique combination of mechanisms and factors including genetic polymorphisms, induction by xenobiotics, regulation by cytokines, hormones and during disease states, as well as sex, age, and others. Multiallelic genetic polymorphisms, which strongly depend on ethnicity, play a major role for the function of CYPs 2D6, 2C19, 2C9, 2B6, 3A5 and 2A6, and lead to distinct pharmacogenetic phenotypes termed as poor, intermediate, extensive, and ultrarapid metabolizers. For these CYPs, the evidence for clinical significance regarding adverse drug reactions (ADRs), drug efficacy and dose requirement is rapidly growing. Polymorphisms in CYPs 1A1, 1A2, 2C8, 2E1, 2J2, and 3A4 are generally less predictive, but new data on CYP3A4 show that predictive variants exist and that additional variants in regulatory genes or in NADPH:cytochrome P450 oxidoreductase (POR) can have an influence. Here we review the recent progress on drug metabolism activity profiles, interindividual variability and regulation of expression, and the functional and clinical impact of genetic variation in drug metabolizing P450s. Copyright © 2013 Elsevier Inc. All rights reserved.
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            Expression of cytochromes P450, conjugating enzymes and nuclear receptors in human hepatoma HepaRG cells.

            Most human hepatocyte cell lines lack a substantial set of liver-specific functions, especially major cytochrome P450 (P450)-related enzyme activities, making them unrepresentative of in vivo hepatocytes. We have used the HepaRG cells, derived from a human hepatocellular carcinoma, which exhibit a high differentiation pattern after 2 weeks at confluency to determine whether they could mimic human hepatocytes for drug metabolism and toxicity studies. We show that when passaged at low density, these cells reversed to an undifferentiated morphology, actively divided, and, after having reached confluency, formed typical hepatocyte-like colonies surrounded by biliary epithelial-like cells. By contrast, when seeded at high density, hepatocyte-like clusters retained their typical differentiated morphology. Transcripts of various nuclear receptors (aryl hydrocarbon receptor, pregnane X receptor, constitutive androstane receptor, peroxisome proliferator-activated receptor alpha), P450s (CYP1A2, 2C9, 2D6, 2E1, 3A4), phase 2 enzymes (UGT1A1, GSTA1, GSTA4, GSTM1), and other liver-specific functions were estimated by reverse transcriptase-quantitative polymerase chain reaction and were found to be expressed, for most of them, at comparable levels in both confluent differentiated and high-density differentiated HepaRG cells and in cultured primary human hepatocytes. For several transcripts, the levels were strongly increased in the presence of 2% dimethyl sulfoxide. Measurement of basal activities of several P450s and their response to prototypical inducers as well as analysis of metabolic profiles and cytotoxicity of several compounds confirmed the functional resemblance of HepaRG cells to primary cultured human hepatocytes. In conclusion, HepaRG cells constitute the first human hepatoma cell line expressing high levels of the major P450s involved in xenobiotic metabolism and represent a reliable surrogate to human hepatocytes for drug metabolism and toxicity studies.
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              Lysine acetylation targets protein complexes and co-regulates major cellular functions.

              Lysine acetylation is a reversible posttranslational modification of proteins and plays a key role in regulating gene expression. Technological limitations have so far prevented a global analysis of lysine acetylation's cellular roles. We used high-resolution mass spectrometry to identify 3600 lysine acetylation sites on 1750 proteins and quantified acetylation changes in response to the deacetylase inhibitors suberoylanilide hydroxamic acid and MS-275. Lysine acetylation preferentially targets large macromolecular complexes involved in diverse cellular processes, such as chromatin remodeling, cell cycle, splicing, nuclear transport, and actin nucleation. Acetylation impaired phosphorylation-dependent interactions of 14-3-3 and regulated the yeast cyclin-dependent kinase Cdc28. Our data demonstrate that the regulatory scope of lysine acetylation is broad and comparable with that of other major posttranslational modifications.

                Author and article information

                Drug Des Devel Ther
                Drug Des Devel Ther
                Drug Design, Development and Therapy
                26 November 2020
                : 14
                : 5251-5258
                [1 ]Department of Clinical Pharmacy, Key Laboratory of Basic Pharmacology of Guizhou Province and School of Pharmacy, Zunyi Medical University , Zunyi 563000, People’s Republic of China
                [2 ]Key Laboratory of Basic Pharmacology of Ministry of Education and Joint International Research Laboratory of Ethnomedicine of Ministry of Education, Zunyi Medical University , Zunyi 563000, People’s Republic of China
                [3 ]Key Laboratory of Clinical Pharmacy of Zunyi City, Zunyi Medical University , Zunyi 563000, People’s Republic of China
                [4 ]Department of Pharmacy, Meitan People’s Hospital , Zunyi 564100, People’s Republic of China
                Author notes
                Correspondence: Fushang Tang Department of Clinical Pharmacy, Key Laboratory of Basic Pharmacology of Guizhou Province and School of Pharmacy , Zunyi563000, People’s Republic of ChinaTel +86 851 2864 2337Fax +86 851 2864 2334 Email fstang@vip.163.com

                These authors contributed equally to this work

                © 2020 Li et al.

                This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License ( http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms ( https://www.dovepress.com/terms.php).

                Page count
                Figures: 5, Tables: 1, References: 47, Pages: 8
                Original Research


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