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1
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Nguyen LTG, Tawfik SM, Jin J, Durwin A, Zhong XB. Impact on efficacy of target reduction of two FDA-approved ASO drugs by intracellular glucose levels in in vitro cell models. MOLECULAR THERAPY. NUCLEIC ACIDS 2025; 36:102487. [PMID: 40104114 PMCID: PMC11919288 DOI: 10.1016/j.omtn.2025.102487] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 10/31/2024] [Accepted: 02/12/2025] [Indexed: 03/20/2025]
Abstract
Antisense oligonucleotides (ASOs) have emerged as a new therapeutic modality for the treatment of both rare and common human diseases. A significant proportion of the patient population that may benefit from ASO therapy may also have common diseases, such as diabetes mellitus. The potential influence of prevalent diseases on the effectiveness of ASO drugs in silencing their target mRNAs remains largely unexplored. The present study utilized in vitro cell models to determine the impact on the efficacy of target reduction of two US Food and Drug Administration (FDA)-approved ASO drugs by intracellular glucose levels. Using inotersen and mipomersen as the FDA-approved ASO model drugs, this study demonstrated that a higher intracellular level of glucose resulted in decreased silencing efficacy of target reduction of inotersen and mipomersen in HepG2 cells. Reducing intracellular glucose levels in HepG2 cells, either by knocking down the glucose transporter GLUT2 or by treating with the antidiabetic drug metformin, reversed the decreased silencing efficacy of inotersen and mipomersen. This study brings to light the first indication about the significant impact of intracellular glucose levels on the silencing efficacy of the FDA-approved ASO drugs in an in vitro model.
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Affiliation(s)
- Le Tra Giang Nguyen
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
| | - Sherouk M Tawfik
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
| | - Jing Jin
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
| | - Andrea Durwin
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
| | - Xiao-Bo Zhong
- Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, Storrs, CT 06269, USA
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2
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Graham RE, Zheng R, Wagner J, Unciti-Broceta A, Hay DC, Forbes SJ, Gadd VL, Carragher NO. Single-cell morphological tracking of cell states to identify small-molecule modulators of liver differentiation. iScience 2025; 28:111871. [PMID: 39995868 PMCID: PMC11848441 DOI: 10.1016/j.isci.2025.111871] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2024] [Revised: 07/24/2024] [Accepted: 01/20/2025] [Indexed: 02/26/2025] Open
Abstract
We have developed a single-cell assay that combines Cell Painting-a morphological profiling assay-with trajectory inference analysis. We have applied this morphological trajectory inference to the bi-potent HepaRG liver progenitor cell line allowing us to track liver cell fate and map small-molecule-induced changes using a morphological atlas of liver cell differentiation. Our overarching goal is to demonstrate the potential of Cell Painting to study biological processes as continuous trajectories at the single-cell level, enhancing resolution and biological understanding. This work has identified small-molecule Src family kinase inhibitors that promote the differentiation of HepaRG cells toward a hepatocyte-like lineage as well as primary human hepatic progenitor cells toward a hepatocyte-like phenotype in vitro. These findings could significantly advance research on liver cell regeneration mechanisms and facilitate the development of cell-based and small-molecule therapies.
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Affiliation(s)
- Rebecca E. Graham
- Centre for Clinical Brain Sciences, University of Edinburgh, Edinburgh EH16 4SB, UK
| | - Runshi Zheng
- Centre for Regenerative Medicine, Institute of Regeneration and Repair, The University of Edinburgh, Edinburgh EH16 4UU, UK
| | - Jesko Wagner
- MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh, Edinburgh EH4 2XU, UK
| | - Asier Unciti-Broceta
- Edinburgh Cancer Research, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK
- Cancer Research UK Scotland Centre, Edinburgh EH4 2XU, UK
| | - David C. Hay
- Centre for Regenerative Medicine, Institute of Regeneration and Repair, The University of Edinburgh, Edinburgh EH16 4UU, UK
| | - Stuart J. Forbes
- Centre for Regenerative Medicine, Institute of Regeneration and Repair, The University of Edinburgh, Edinburgh EH16 4UU, UK
| | - Victoria L. Gadd
- Centre for Regenerative Medicine, Institute of Regeneration and Repair, The University of Edinburgh, Edinburgh EH16 4UU, UK
| | - Neil O. Carragher
- Edinburgh Cancer Research, Institute of Genetics and Cancer, University of Edinburgh, Western General Hospital, Edinburgh EH4 2XU, UK
- Cancer Research UK Scotland Centre, Edinburgh EH4 2XU, UK
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3
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Angendohr C, Missing L, Ehlting C, Wolf SD, Lang KS, Vucur M, Luedde T, Bode JG. Interleukin 1 β suppresses bile acid-induced BSEP expression via a CXCR2-dependent feedback mechanism. PLoS One 2024; 19:e0315243. [PMID: 39680527 DOI: 10.1371/journal.pone.0315243] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2024] [Accepted: 11/22/2024] [Indexed: 12/18/2024] Open
Abstract
Inflammation-induced cholestasis is a common problem in septic patients and results from cytokine-mediated inhibition of bile acid export including impaired expression of the bile salt export pump (BSEP) with a consecutive increase in intracellular bile acids mediating cell damage. The present study focuses on the mechanisms by which interleukin 1 β (IL-1β), as a critical mediator of sepsis-induced cholestasis, controls the expression of BSEP in hepatocytes. Notably, the treatment of hepatocytes with IL-1β leads to the upregulation of a broad chemokine pattern. Thereby, the IL-1β -induced expression of in particular the CXCR2 ligands CXCL1 and 2 is further enhanced by bile acids, whereas the FXR-mediated upregulation of BSEP induced by bile acids is inhibited by IL-1β. In this context, it is interesting to note that inhibitor studies indicate that IL-1β mediates its inhibitory effects on bile acid-induced expression of BSEP indirectly via CXCR2 ligands. Consistently, inhibition of CXCR2 with the inhibitor SB225002 significantly attenuated of the inhibitory effect of IL-1β on BSEP expression. These data suggest that part of the cholestasis-inducing effect of IL-1β is mediated via a CXCR2-dependent feedback mechanism.
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Affiliation(s)
- Carolin Angendohr
- Faculty of Medicine & Düsseldorf University Hospital, Department of Gastroenterology, Hepatology and Infectious Disease, Heinrich-Heine-University, Düsseldorf, Germany
| | - Leah Missing
- Faculty of Medicine & Düsseldorf University Hospital, Department of Gastroenterology, Hepatology and Infectious Disease, Heinrich-Heine-University, Düsseldorf, Germany
| | - Christian Ehlting
- Faculty of Medicine & Düsseldorf University Hospital, Department of Gastroenterology, Hepatology and Infectious Disease, Heinrich-Heine-University, Düsseldorf, Germany
| | - Stephanie D Wolf
- Faculty of Medicine & Düsseldorf University Hospital, Department of Gastroenterology, Hepatology and Infectious Disease, Heinrich-Heine-University, Düsseldorf, Germany
| | - Karl S Lang
- Department of Immunology, University of Essen, Essen, Germany
| | - Mihael Vucur
- Faculty of Medicine & Düsseldorf University Hospital, Department of Gastroenterology, Hepatology and Infectious Disease, Heinrich-Heine-University, Düsseldorf, Germany
| | - Tom Luedde
- Faculty of Medicine & Düsseldorf University Hospital, Department of Gastroenterology, Hepatology and Infectious Disease, Heinrich-Heine-University, Düsseldorf, Germany
| | - Johannes G Bode
- Faculty of Medicine & Düsseldorf University Hospital, Department of Gastroenterology, Hepatology and Infectious Disease, Heinrich-Heine-University, Düsseldorf, Germany
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4
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Makris G, Veit L, Rüfenacht V, Klassa S, Zürcher N, Matsumoto S, Poms M, Häberle J. Expression and function of the urea cycle in widely-used hepatic cellular models. J Inherit Metab Dis 2024; 47:1228-1238. [PMID: 38192032 DOI: 10.1002/jimd.12701] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2023] [Revised: 11/30/2023] [Accepted: 12/04/2023] [Indexed: 01/10/2024]
Abstract
The group of rare metabolic defects termed urea cycle disorders (UCDs) occur within the ammonia elimination pathway and lead to significant neurocognitive sequelae for patients surviving decompensation episodes. Besides orthotopic liver transplantation, curative options are lacking for UCDs, with dietary management being the gold clinical standard. Novel therapeutic approaches are essential for UCDs; however, such effort presupposes preclinical testing in cellular models that effectively capture disease manifestation. Several cellular and animal models exist and aim to recapitulate the broad phenotypic spectrum of UCDs; however, the majority of those lack extensive molecular and biochemical characterization. The development of cellular models is emerging since animal models are extremely time and cost consuming, and subject to ethical considerations, including the 3R principle that endorses animal welfare over unchecked preclinical testing. The aim of this study was to compare the extent of expression and functionality of the urea cycle in two commercial hepatoma-derived cell lines, induced pluripotent stem cell hepatocytes (iPSC-Heps), primary human hepatocytes (PHHs) and human liver cell preparations. Using immunoblotting, immunocytochemistry, and stable isotope tracing of the urea cycle metabolites, we identified that the hepatoma-derived, 2-week differentiated HepaRG cells are urea cycle proficient and behave as cellular alternatives to PHHs. Furthermore, HepaRG cells were superior to iPSC-Heps, which are known to exhibit batch-to-batch variabilities in terms of hepatic maturity and enzyme expression. Finally, HepG2 cells lack the urea cycle enzymes ornithine transcarbamylase and arginase 1, the transporter ORNT1, which limits their suitability as model for the study of UCDs.
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Affiliation(s)
- Georgios Makris
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
- Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland
| | - Lara Veit
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Véronique Rüfenacht
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Sven Klassa
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Nadia Zürcher
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
| | - Shirou Matsumoto
- Department of Pediatrics, Graduate School of Medical Sciences, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
| | - Martin Poms
- Division of Clinical Chemistry and Biochemistry, University Children's Hospital Zurich, Zurich, Switzerland
| | - Johannes Häberle
- Division of Metabolism and Children's Research Center, University Children's Hospital Zurich, Zurich, Switzerland
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5
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Li Y, Bowling AJ, Lehman A, Johnson K, Pence HE, Breitweiser LA, Sherer E, LaRocca J, Chen W. High-Throughput Image-Based Assay for Identifying In Vitro Hepatocyte Microtubule Disruption. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2024; 72:21804-21819. [PMID: 39312225 DOI: 10.1021/acs.jafc.4c04969] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/03/2024]
Abstract
Disruption of microtubule stability in mammalian cells may lead to genotoxicity and carcinogenesis. The ability to screen for microtubule destabilization or stabilization is therefore a useful and efficient approach to aid in the design of molecules that are safe for human health. In this study, we developed a high-throughput 384-well assay combining immunocytochemistry with high-content imaging to assess microtubule disruption in the metabolically competent human liver cell line: HepaRG. To enhance analysis throughput, we implemented a supervised machine learning approach using a curated training library of 180 compounds. A majority voting ensemble of eight machine learning classifiers was employed for predicting microtubule disruptions. Our prediction model achieved over 99.0% accuracy and a 98.4% F1 score, which reflects the balance between precision and recall for in-sample validation and 93.5% accuracy and a 94.3% F1 score for out-of-sample validation. This automated image-based testing can provide a simple, high-throughput screening method for early stage discovery compounds to reduce the potential risk of genotoxicity for crop protection product development.
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Affiliation(s)
- Yang Li
- Corteva Agriscience, Indianapolis, Indiana 46268, United States
| | | | - Audrey Lehman
- Corteva Agriscience, Indianapolis, Indiana 46268, United States
| | | | - Heather E Pence
- Corteva Agriscience, Indianapolis, Indiana 46268, United States
| | | | - Eric Sherer
- Corteva Agriscience, Indianapolis, Indiana 46268, United States
| | - Jessica LaRocca
- Corteva Agriscience, Indianapolis, Indiana 46268, United States
| | - Wei Chen
- Corteva Agriscience, Indianapolis, Indiana 46268, United States
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6
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Bianchi E, Costa E, Harrill J, Deford P, LaRocca J, Chen W, Sutake Z, Lehman A, Pappas-Garton A, Sherer E, Moreillon C, Sriram S, Dhroso A, Johnson K. Discovery Phase Agrochemical Predictive Safety Assessment Using High Content In Vitro Data to Estimate an In Vivo Toxicity Point of Departure. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2024. [PMID: 39033510 DOI: 10.1021/acs.jafc.4c03094] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/23/2024]
Abstract
Utilization of in vitro (cellular) techniques, like Cell Painting and transcriptomics, could provide powerful tools for agrochemical candidate sorting and selection in the discovery process. However, using these models generates challenges translating in vitro concentrations to the corresponding in vivo exposures. Physiologically based pharmacokinetic (PBPK) modeling provides a framework for quantitative in vitro to in vivo extrapolation (IVIVE). We tested whether in vivo (rat liver) transcriptomic and apical points of departure (PODs) could be accurately predicted from in vitro (rat hepatocyte or human HepaRG) transcriptomic PODs or HepaRG Cell Painting PODs using PBPK modeling. We compared two PBPK models, the ADMET predictor and the httk R package, and found httk to predict the in vivo PODs more accurately. Our findings suggest that a rat liver apical and transcriptomic POD can be estimated utilizing a combination of in vitro transcriptome-based PODs coupled with PBPK modeling for IVIVE. Thus, high content in vitro data can be translated with modest accuracy to in vivo models of ultimate regulatory importance to help select agrochemical analogs in early stage discovery program.
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Affiliation(s)
- Enrica Bianchi
- Corteva Agriscience, Indianapolis ,Indiana 46268, United States
| | | | - Joshua Harrill
- Center for Computational Toxicology and Exposure, United States Environmental Protection Agency, Research Triangle Park ,North Carolina 27709, United States
| | - Paul Deford
- Corteva Agriscience, Indianapolis ,Indiana 46268, United States
| | - Jessica LaRocca
- Corteva Agriscience, Indianapolis ,Indiana 46268, United States
| | - Wei Chen
- Corteva Agriscience, Indianapolis ,Indiana 46268, United States
| | - Zachary Sutake
- Corteva Agriscience, Indianapolis ,Indiana 46268, United States
| | - Audrey Lehman
- Corteva Agriscience, Indianapolis ,Indiana 46268, United States
| | | | - Eric Sherer
- Corteva Agriscience, Indianapolis ,Indiana 46268, United States
| | | | | | - Andi Dhroso
- Corteva Agriscience, Indianapolis ,Indiana 46268, United States
| | - Kamin Johnson
- Corteva Agriscience, Indianapolis ,Indiana 46268, United States
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7
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Guo X, Xu H, Seo JE. Application of HepaRG cells for genotoxicity assessment: a review. JOURNAL OF ENVIRONMENTAL SCIENCE AND HEALTH. PART C, TOXICOLOGY AND CARCINOGENESIS 2024; 42:214-237. [PMID: 38566478 DOI: 10.1080/26896583.2024.2331956] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 04/04/2024]
Abstract
There has been growing interest in the use of human-derived metabolically competent cells for genotoxicity testing. The HepaRG cell line is considered one of the most promising cell models because it is TP53-proficient and retains many characteristics of primary human hepatocytes. In recent years, HepaRG cells, cultured in both a traditional two-dimensional (2D) format and as more advanced in-vivo-like 3D spheroids, have been employed in assays that measure different types of genetic toxicity endpoints, including DNA damage, mutations, and chromosomal damage. This review summarizes published studies that have used HepaRG cells for genotoxicity assessment, including cell model evaluation studies and risk assessment for various compounds. Both 2D and 3D HepaRG models can be adapted to several high-throughput genotoxicity assays, generating a large number of data points that facilitate quantitative benchmark concentration modeling. With further validation, HepaRG cells could serve as a unique, human-based new alternative methodology for in vitro genotoxicity testing.
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Affiliation(s)
- Xiaoqing Guo
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR, USA
| | - Hannah Xu
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR, USA
| | - Ji-Eun Seo
- Division of Genetic and Molecular Toxicology, National Center for Toxicological Research, Jefferson, AR, USA
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8
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Kuhn J, McDonald A, Mongoin C, Anderson G, Lafeuillade G, Mitchell S, Elfick APD, Bagnaninchi PO, Yiu HHP, Nelson LJ. Non-invasive methods of monitoring Fe 3O 4 magnetic nanoparticle toxicity in human liver HepaRG cells using impedance biosensing and Coherent anti-Stokes Raman spectroscopic (CARS) microscopy. Toxicol Lett 2024; 394:92-101. [PMID: 38428546 DOI: 10.1016/j.toxlet.2024.02.010] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2023] [Revised: 02/09/2024] [Accepted: 02/21/2024] [Indexed: 03/03/2024]
Abstract
Functionalized nanoparticles have been developed for use in nanomedicines for treating life threatening diseases including various cancers. To ensure safe use of these new nanoscale reagents, various assays for biocompatibility or cytotoxicity in vitro using cell lines often serve as preliminary assessments prior to in vivo animal testing. However, many of these assays were designed for soluble, colourless materials and may not be suitable for coloured, non-transparent nanoparticles. Moreover, cell lines are not always representative of mammalian organs in vivo. In this work, we use non-invasive impedance sensing methods with organotypic human liver HepaRG cells as a model to test the toxicity of PEG-Fe3O4 magnetic nanoparticles. We also use Coherent anti-Stokes Raman Spectroscopic (CARS) microscopy to monitor the formation of lipid droplets as a parameter to the adverse effect on the HepaRG cell model. The results were also compared with two commercial testing kits (PrestoBlue and ATP) for cytotoxicity. The results suggested that the HepaRG cell model can be a more realistic model than commercial cell lines while use of impedance monitoring of Fe3O4 nanoparticles circumventing the uncertainties due to colour assays. These methods can play important roles for scientists driving towards the 3Rs principle - Replacement, Reduction and Refinement.
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Affiliation(s)
- Joel Kuhn
- Institute of Chemical Sciences, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, Scotland EH14 4AS, UK
| | - Alison McDonald
- Institute for Bioengineering, School of Engineering, The University of Edinburgh, King's Buildings, Edinburgh EH9 3DW
| | - Cyril Mongoin
- Institute for Bioengineering, School of Engineering, The University of Edinburgh, King's Buildings, Edinburgh EH9 3DW
| | - Graham Anderson
- Centre for Regenerative Medicine. Institute for Regeneration and Repair, The University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, UK
| | - Guillemette Lafeuillade
- Institute for Bioengineering, School of Engineering, The University of Edinburgh, King's Buildings, Edinburgh EH9 3DW
| | - Stephen Mitchell
- School of Biological Sciences, The Daniel Rutherford Building, Max Born Crescent, Edinburgh EH9 3BF, UK
| | - Alistair P D Elfick
- Institute for Bioengineering, School of Engineering, The University of Edinburgh, King's Buildings, Edinburgh EH9 3DW
| | - Pierre O Bagnaninchi
- Centre for Regenerative Medicine. Institute for Regeneration and Repair, The University of Edinburgh, Edinburgh BioQuarter, 5 Little France Drive, Edinburgh EH16 4UU, UK
| | - Humphrey H P Yiu
- Institute of Chemical Sciences, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, Scotland EH14 4AS, UK.
| | - Leonard J Nelson
- Institute for Bioengineering, School of Engineering, The University of Edinburgh, King's Buildings, Edinburgh EH9 3DW.
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9
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Huber AD, Poudel S, Wu J, Miller DJ, Lin W, Yang L, Bwayi MN, Rimmer MA, Gee RRF, Seetharaman J, Chai SC, Chen T. A bromodomain-independent mechanism of gene regulation by the BET inhibitor JQ1: direct activation of nuclear receptor PXR. Nucleic Acids Res 2024; 52:1661-1676. [PMID: 38084912 PMCID: PMC10899790 DOI: 10.1093/nar/gkad1175] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/03/2023] [Revised: 11/17/2023] [Accepted: 11/23/2023] [Indexed: 02/29/2024] Open
Abstract
Bromodomain and extraterminal (BET) proteins are extensively studied in multiple pathologies, including cancer. BET proteins modulate transcription of various genes, including those synonymous with cancer, such as MYC. Thus, BET inhibitors are a major area of drug development efforts. (+)-JQ1 (JQ1) is the prototype inhibitor and is a common tool to probe BET functions. While showing therapeutic promise, JQ1 is not clinically usable, partly due to metabolic instability. Here, we show that JQ1 and the BET-inactive (-)-JQ1 are agonists of pregnane X receptor (PXR), a nuclear receptor that transcriptionally regulates genes encoding drug-metabolizing enzymes such as CYP3A4, which was previously shown to oxidize JQ1. A PXR-JQ1 co-crystal structure identified JQ1's tert-butyl moiety as a PXR anchor and explains binding by (-)-JQ1. Analogs differing at the tert-butyl lost PXR binding, validating our structural findings. Evaluation in liver cell models revealed both PXR-dependent and PXR-independent modulation of CYP3A4 expression by BET inhibitors. We have characterized a non-BET JQ1 target, a mechanism of physiological JQ1 instability, a biological function of (-)-JQ1, and BET-dependent transcriptional regulation of drug metabolism genes.
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Affiliation(s)
- Andrew D Huber
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Shyaron Poudel
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Jing Wu
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Darcie J Miller
- Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Wenwei Lin
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Lei Yang
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Monicah N Bwayi
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Mary Ashley Rimmer
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Rebecca R Florke Gee
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
- Graduate School of Biomedical Sciences, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Jayaraman Seetharaman
- Department of Structural Biology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Sergio C Chai
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
| | - Taosheng Chen
- Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105, USA
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10
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Hanaoka K, Ikeno T, Iwaki S, Deguchi S, Takayama K, Mizuguchi H, Tao F, Kojima N, Ohno H, Sasaki E, Komatsu T, Ueno T, Maeda K, Kusuhara H, Urano Y. A general fluorescence off/on strategy for fluorogenic probes: Steric repulsion-induced twisted intramolecular charge transfer (sr-TICT). SCIENCE ADVANCES 2024; 10:eadi8847. [PMID: 38363840 PMCID: PMC10871538 DOI: 10.1126/sciadv.adi8847] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/23/2023] [Accepted: 01/17/2024] [Indexed: 02/18/2024]
Abstract
Various control strategies are available for building fluorogenic probes to visualize biological events in terms of a fluorescence change. Here, we performed the time-dependent density functional theory (TD-DFT) computational analysis of the twisted intramolecular charge transfer (TICT) process in rhodamine dyes. On the basis of the results, we designed and synthesized a series of rhodamine dyes and established a fluorescence quenching strategy that we call steric repulsion-induced TICT (sr-TICT), in which the fluorescence quenching process is greatly accelerated by simple intramolecular twisting. As proof of concept of this design strategy, we used it to develop a fluorogenic probe, 2-Me PeER (pentyloxyethylrhodamine), for the N-dealkylation activity of CYP3A4. We applied 2-Me PeER for CYP3A4 activity-based fluorescence-activated cell sorting (FACS), providing access to homogeneous, highly functional human-induced pluripotent stem cell (hiPSC)-derived hepatocytes and intestinal epithelial cells. Our results suggest that sr-TICT represents a general fluorescence control method for fluorogenic probes.
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Affiliation(s)
- Kenjiro Hanaoka
- Graduate School of Pharmaceutical Sciences, Keio University, 1-5-30 Shibakoen, Minoto-ku, Tokyo 105-8512, Japan
| | - Takayuki Ikeno
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Shimpei Iwaki
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Sayaka Deguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Kazuo Takayama
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
| | - Hiroyuki Mizuguchi
- Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan
- Integrated Frontier Research for Medical Science Division, Institute for Open and Transdisciplinary Research Initiatives, Osaka University, Osaka 565-0871, Japan
- National Institutes of Biomedical Innovation, Health and Nutrition, Osaka 567-0085, Japan
| | - Fumiya Tao
- Department of Life and Environmental System Science, Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
| | - Nobuhiko Kojima
- Department of Life and Environmental System Science, Graduate School of Nanobioscience, Yokohama City University, 22-2 Seto, Kanazawa-ku, Yokohama 236-0027, Japan
| | - Hisashi Ohno
- Graduate School of Pharmaceutical Sciences, Keio University, 1-5-30 Shibakoen, Minoto-ku, Tokyo 105-8512, Japan
| | - Eita Sasaki
- Graduate School of Pharmaceutical Sciences, Keio University, 1-5-30 Shibakoen, Minoto-ku, Tokyo 105-8512, Japan
| | - Toru Komatsu
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Tasuku Ueno
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Kazuya Maeda
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Hiroyuki Kusuhara
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
| | - Yasuteru Urano
- Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
- Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
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11
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Hirukawa K, Yagi H, Kuroda K, Watanabe M, Nishi K, Nagata S, Abe Y, Kitago M, Adachi S, Sudo R, Kitagawa Y. Novel approach for reconstruction of the three-dimensional biliary system in decellularized liver scaffold using hepatocyte progenitors. PLoS One 2024; 19:e0297285. [PMID: 38359035 PMCID: PMC10868823 DOI: 10.1371/journal.pone.0297285] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/07/2023] [Accepted: 01/02/2024] [Indexed: 02/17/2024] Open
Abstract
Reconstruction of the biliary system is indispensable for the regeneration of transplantable liver grafts. Here, we report the establishment of the first continuous three-dimensional biliary system scaffold for bile acid excretion using a novel method. We confirmed the preservation of the liver-derived extracellular matrix distribution in the scaffold. In addition, hepatocyte progenitors decellularized via the bile duct by slow-speed perfusion differentiated into hepatocyte- and cholangiocyte-like cells, mimicking hepatic cords and bile ducts, respectively. Furthermore, qRT-PCR demonstrated increased ALB, BSEP, and AQP8 expression, revealing bile canaliculi- and bile duct-specific genetic patterns. Therefore, we concluded that locally preserved extracellular matrices in the scaffold stimulated hepatic progenitors and provided efficient differentiation, as well as regeneration of a three-dimensional continuous biliary system from hepatic cords through bile ducts. These findings suggest that organ-derived scaffolds can be utilized for the efficient reconstruction of functional biliary systems.
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Affiliation(s)
- Kazuya Hirukawa
- Department of Surgery, Keio University School of Medicine, Shinanomachi, Shinjuku, Japan
| | - Hiroshi Yagi
- Department of Surgery, Keio University School of Medicine, Shinanomachi, Shinjuku, Japan
| | - Kohei Kuroda
- Department of Surgery, Keio University School of Medicine, Shinanomachi, Shinjuku, Japan
| | - Masafumi Watanabe
- Institute of Materials Science and Technology (E308), Technische Universität Wien, Vienna, Austria
- Department of System Design Engineering, Keio University, Kohoku-ku, Yokohama, Japan
| | - Kotaro Nishi
- Department of Surgery, Keio University School of Medicine, Shinanomachi, Shinjuku, Japan
| | - Shogo Nagata
- Department of Surgery, Keio University School of Medicine, Shinanomachi, Shinjuku, Japan
| | - Yuta Abe
- Department of Surgery, Keio University School of Medicine, Shinanomachi, Shinjuku, Japan
| | - Minoru Kitago
- Department of Surgery, Keio University School of Medicine, Shinanomachi, Shinjuku, Japan
| | - Shungo Adachi
- Molecular Profiling Research Center for Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan
| | - Ryo Sudo
- Department of System Design Engineering, Keio University, Kohoku-ku, Yokohama, Japan
| | - Yuko Kitagawa
- Department of Surgery, Keio University School of Medicine, Shinanomachi, Shinjuku, Japan
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12
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Saliba-Gustafsson P, Justesen JM, Ranta A, Sharma D, Bielczyk-Maczynska E, Li J, Najmi LA, Apodaka M, Aspichueta P, Björck HM, Eriksson P, Franco-Cereceda A, Gloudemans M, Mujica E, den Hoed M, Assimes TL, Quertermous T, Carcamo-Orive I, Park CY, Knowles JW. A functional genomic framework to elucidate novel causal non-alcoholic fatty liver disease genes. MEDRXIV : THE PREPRINT SERVER FOR HEALTH SCIENCES 2024:2024.02.03.24302258. [PMID: 38352379 PMCID: PMC10863038 DOI: 10.1101/2024.02.03.24302258] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 02/21/2024]
Abstract
Background & Aims Non-alcoholic fatty liver disease (NAFLD) is the most prevalent chronic liver pathology in western countries, with serious public health consequences. Efforts to identify causal genes for NAFLD have been hampered by the relative paucity of human data from gold-standard magnetic resonance quantification of hepatic fat. To overcome insufficient sample size, genome-wide association studies using NAFLD surrogate phenotypes have been used, but only a small number of loci have been identified to date. In this study, we combined GWAS of NAFLD composite surrogate phenotypes with genetic colocalization studies followed by functional in vitro screens to identify bona fide causal genes for NAFLD. Approach & Results We used the UK Biobank to explore the associations of our novel NAFLD score, and genetic colocalization to prioritize putative causal genes for in vitro validation. We created a functional genomic framework to study NAFLD genes in vitro using CRISPRi. Our data identify VKORC1, TNKS, LYPLAL1 and GPAM as regulators of lipid accumulation in hepatocytes and suggest the involvement of VKORC1 in the lipid storage related to the development of NAFLD. Conclusions Complementary genetic and genomic approaches are useful for the identification of NAFLD genes. Our data supports VKORC1 as a bona fide NAFLD gene. We have established a functional genomic framework to study at scale putative novel NAFLD genes from human genetic association studies.
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Affiliation(s)
- Peter Saliba-Gustafsson
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- CardioMetabolic Unit at the Department of Medicine, Huddinge, Karolinska Institutet, Stockholm, Sweden
- Stanford Diabetes Research Center, Stanford, CA, USA
- Stanford Cardiovascular Institute, Stanford University School of Medicine, CA, USA
| | - Johanne M. Justesen
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark
| | - Amanda Ranta
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- Stanford Diabetes Research Center, Stanford, CA, USA
- Stanford Cardiovascular Institute, Stanford University School of Medicine, CA, USA
| | - Disha Sharma
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- Stanford Cardiovascular Institute, Stanford University School of Medicine, CA, USA
| | - Ewa Bielczyk-Maczynska
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- Stanford Diabetes Research Center, Stanford, CA, USA
- Stanford Cardiovascular Institute, Stanford University School of Medicine, CA, USA
- The Hormel Institute, University of Minnesota, MN, USA
| | - Jiehan Li
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- Stanford Diabetes Research Center, Stanford, CA, USA
- Stanford Cardiovascular Institute, Stanford University School of Medicine, CA, USA
| | - Laeya A. Najmi
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- Stanford Diabetes Research Center, Stanford, CA, USA
- Stanford Cardiovascular Institute, Stanford University School of Medicine, CA, USA
| | - Maider Apodaka
- University of the Basque Country (UPV/EHU), Faculty of Medicine and Nursing, Department of Physiology, Leioa, Spain
| | - Patricia Aspichueta
- University of the Basque Country (UPV/EHU), Faculty of Medicine and Nursing, Department of Physiology, Leioa, Spain
- National Institute for the Study of Liver and Gastrointestinal Diseases (CIBERehd, Instituto de Salud Carlos III)
| | - Hanna M. Björck
- Division of Cardiovascular Medicine, Centre for Molecular Medicine, Department of Medicine, Solna, Karolinska Inistitutet, Stockholm, Karolinska University Hospital, Solna, Sweden
| | - Per Eriksson
- Division of Cardiovascular Medicine, Centre for Molecular Medicine, Department of Medicine, Solna, Karolinska Inistitutet, Stockholm, Karolinska University Hospital, Solna, Sweden
| | | | - Mike Gloudemans
- Department of Pathology, Stanford University School of Medicine, CA, USA
| | - Endrina Mujica
- Department of Immunology, Genetics and Pathology, Uppsala University, Sweden
| | - Marcel den Hoed
- Department of Immunology, Genetics and Pathology, Uppsala University, Sweden
| | - Themistocles L. Assimes
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- VA Palo Alto Health Care System, Palo Alto CA, USA
| | - Thomas Quertermous
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- Novo Nordisk Foundation Center for Basic Metabolic Research, University of Copenhagen, Denmark
| | - Ivan Carcamo-Orive
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- IKERBASQUE, Basque Foundation for Science, Bilbao, Spain
| | - Chong Y. Park
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
| | - Joshua W. Knowles
- Department of Medicine, Division of Cardiovascular Medicine and Cardiovascular Institute, Stanford University, Stanford, CA, USA
- Stanford Diabetes Research Center, Stanford, CA, USA
- Stanford Cardiovascular Institute, Stanford University School of Medicine, CA, USA
- Stanford Prevention Research Center, Stanford, CA, USA
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13
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Babu S, Ranajit SK, Pattnaik G, Ghosh G, Rath G, Kar B. An Insight into Different Experimental Models used for Hepatoprotective Studies: A Review. Curr Drug Discov Technol 2024; 21:e191223224660. [PMID: 39206705 DOI: 10.2174/0115701638278844231214115102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/24/2023] [Revised: 11/13/2023] [Accepted: 11/22/2023] [Indexed: 09/04/2024]
Abstract
Numerous factors, including exposure to harmful substances, drinking too much alcohol, contracting certain hepatitis serotypes, and using specific medicines, contribute to the development of liver illnesses. Lipid peroxidation and other forms of oxidative stress are the main mechanisms by which hepatotoxic substances harm liver cells. Pathological changes in the liver include a rise in the levels of blood serum, a decrease in antioxidant enzymes, as well as the formation of free radical radicals. It is necessary to find pharmaceutical alternatives to treat liver diseases to increase their efficacy and decrease their toxicity. For the development of new therapeutic medications, a greater knowledge of primary mechanisms is required. In order to mimic human liver diseases, animal models are developed. Animal models have been used for several decades to study the pathogenesis of liver disorders and related toxicities. For many years, animal models have been utilized to investigate the pathophysiology of liver illness and associated toxicity. The animal models are created to imitate human hepatic disorders. This review enlisted numerous hepatic damage in vitro and in vivo models using various toxicants, their probable biochemical pathways and numerous metabolic pathways via oxidative stressors, different serum biomarkers enzymes are discussed, which will help to identify the most accurate and suitable model to test any plant preparations to check and evaluate their hepatoprotective properties.
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Affiliation(s)
- Sucharita Babu
- School of Pharmacy and Life Sciences, Centurion University of Technology and Management, Bhubaneswar, 751050, India
| | - Santosh K Ranajit
- School of Pharmacy and Life Sciences, Centurion University of Technology and Management, Bhubaneswar, 751050, India
| | - Gurudutta Pattnaik
- School of Pharmacy and Life Sciences, Centurion University of Technology and Management, Bhubaneswar, 751050, India
| | - Goutam Ghosh
- School of Pharmaceutical Sciences, Siksha O Anusandhan Deemed to be University, Bhubaneswar, 751030, India
| | - Goutam Rath
- School of Pharmaceutical Sciences, Siksha O Anusandhan Deemed to be University, Bhubaneswar, 751030, India
| | - Biswakanth Kar
- School of Pharmaceutical Sciences, Siksha O Anusandhan Deemed to be University, Bhubaneswar, 751030, India
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14
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Settivari RS, Martini A, Wijeyesakere S, Toltin A, LeBaron MJ. Application of Evolving New Approach Methodologies for Chemical Safety Assessment. A COMPREHENSIVE GUIDE TO TOXICOLOGY IN NONCLINICAL DRUG DEVELOPMENT 2024:977-1015. [DOI: 10.1016/b978-0-323-85704-8.00026-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2025]
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15
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Dellbrügge F, Jesse LD, Medyukhina A, Liu N, Neugebauer S, Freißmuth M, Höppener S, Figge MT, Morrison H, Riecken LB, Press AT. Contribution of radixin and ezrin to the maintenance of hepatocytes' excretory function in health and disease. Heliyon 2023; 9:e21009. [PMID: 37928027 PMCID: PMC10623174 DOI: 10.1016/j.heliyon.2023.e21009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2023] [Revised: 09/29/2023] [Accepted: 10/12/2023] [Indexed: 11/07/2023] Open
Abstract
Background & aims Excretory liver failure is frequently associated with poor prognosis in critically ill patients. It is characterized by the loss of canalicular membrane export pumps at the hepatocyte membrane. The membrane export pump Multidrug resistant-associated protein (MRP) 2 is pivotal in hepatocytes for brushed membrane morphology and transport of various metabolites. In addition, MRP2 anchoring proteins of the Ezrin/Radixin/Moesin (ERM) family are crucial for the correct MRP2 location, integration, and function in different tissues. In hepatocytes, altered ERM signaling is elementary for developing excretory liver failure. Methods Polarized human HepaRG cells, primary human hepatocytes, and hepatocyte-specific Ezrin knockout mice are employed to investigate ERM expression and function in health and the bile duct ligation model of obstructive cholestasis. Results ERM-scaffolding protein Ezrin has no relevant function in maintaining the canalicular structure in hepatocytes during health and disease. Conclusions Homeostasis of the canalicular pole in hepatocytes is maintained exclusively by Radixin but not Ezrin, and Radixin dysfunction promotes cholestasis.
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Affiliation(s)
- Friederike Dellbrügge
- Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Am Klinikum 1, 07740, Jena, Germany
- Center for Sepsis Control and Care, Jena University Hospital, Am Klinikum 1, 07740, Jena, Germany
| | - Lena D. Jesse
- Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Am Klinikum 1, 07740, Jena, Germany
- Center for Sepsis Control and Care, Jena University Hospital, Am Klinikum 1, 07740, Jena, Germany
| | - Anna Medyukhina
- Research Group Applied Systems Biology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Beutenbergstraße 11a, 07745, Jena, Germany
| | - Na Liu
- Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Am Klinikum 1, 07740, Jena, Germany
| | - Sophie Neugebauer
- Department of Clinical Chemistry and Laboratory Diagnostics, Jena University Hospital, Am Klinikum 1, 07740, Jena, Germany
| | - Markus Freißmuth
- Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Am Klinikum 1, 07740, Jena, Germany
| | - Stephanie Höppener
- Laboratory of Organic and Macromolecular Chemistry (IOMC), Friedrich-Schiller University, Fürstengraben 1, 07737, Jena, Germany
| | - Marc T. Figge
- Research Group Applied Systems Biology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Beutenbergstraße 11a, 07745, Jena, Germany
- Faculty of Biological Sciences, Friedrich-Schiller University, Fürstengraben 1, 07737, Jena, Germany
| | - Helen Morrison
- Faculty of Biological Sciences, Friedrich-Schiller University, Fürstengraben 1, 07737, Jena, Germany
- Leibniz Institute on Aging, Beutenbergstraße 11, 07745, Jena, Germany
| | - Lars B. Riecken
- Leibniz Institute on Aging, Beutenbergstraße 11, 07745, Jena, Germany
| | - Adrian T. Press
- Department of Anesthesiology and Intensive Care Medicine, Jena University Hospital, Am Klinikum 1, 07740, Jena, Germany
- Center for Sepsis Control and Care, Jena University Hospital, Am Klinikum 1, 07740, Jena, Germany
- Medical Faculty, Friedrich-Schiller University, Fürstengraben 1, 07737, Jena, Germany
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16
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Sun L, Wang Y, Zhang S, Yang H, Mao Y. 3D bioprinted liver tissue and disease models: Current advances and future perspectives. BIOMATERIALS ADVANCES 2023; 152:213499. [PMID: 37295133 DOI: 10.1016/j.bioadv.2023.213499] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/27/2022] [Revised: 02/23/2023] [Accepted: 06/02/2023] [Indexed: 06/12/2023]
Abstract
Three-dimensional (3D) bioprinting is a promising technology for fabricating complex tissue constructs with biomimetic biological functions and stable mechanical properties. In this review, the characteristics of different bioprinting technologies and materials are compared, and development in strategies for bioprinting normal and diseased hepatic tissue are summarized. In particular, features of bioprinting and other bio-fabrication strategies, such as organoids and spheroids are compared to demonstrate the strengths and weaknesses of 3D printing technology. Directions and suggestions, such as vascularization and primary human hepatocyte culture, are provided for the future development of 3D bioprinting.
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Affiliation(s)
- Lejia Sun
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Dongcheng, Beijing, 100730, China; Department of General Surgery, The First affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China
| | - Yinhan Wang
- Peking Union Medical College (PUMC), Chinese Academy of Medical Sciences & PUMC, Dongcheng, Beijing 100730, China
| | - Shuquan Zhang
- Peking Union Medical College (PUMC), Chinese Academy of Medical Sciences & PUMC, Dongcheng, Beijing 100730, China
| | - Huayu Yang
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Dongcheng, Beijing, 100730, China.
| | - Yilei Mao
- Department of Liver Surgery, Peking Union Medical College (PUMC) Hospital, PUMC & Chinese Academy of Medical Sciences, Dongcheng, Beijing, 100730, China.
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17
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Ivanova ON, Krasnov GS, Snezhkina AV, Kudryavtseva AV, Fedorov VS, Zakirova NF, Golikov MV, Kochetkov SN, Bartosch B, Valuev-Elliston VT, Ivanov AV. Transcriptome Analysis of Redox Systems and Polyamine Metabolic Pathway in Hepatoma and Non-Tumor Hepatocyte-like Cells. Biomolecules 2023; 13:714. [PMID: 37189460 PMCID: PMC10136275 DOI: 10.3390/biom13040714] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2023] [Revised: 04/10/2023] [Accepted: 04/19/2023] [Indexed: 05/17/2023] Open
Abstract
Reactive oxygen species (ROS) play a major role in the regulation of various processes in the cell. The increase in their production is a factor contributing to the development of numerous pathologies, including inflammation, fibrosis, and cancer. Accordingly, the study of ROS production and neutralization, as well as redox-dependent processes and the post-translational modifications of proteins, is warranted. Here, we present a transcriptomic analysis of the gene expression of various redox systems and related metabolic processes, such as polyamine and proline metabolism and the urea cycle in Huh7.5 hepatoma cells and the HepaRG liver progenitor cell line, that are widely used in hepatitis research. In addition, changes in response to the activation of polyamine catabolism that contribute to oxidative stress were studied. In particular, differences in the gene expression of various ROS-producing and ROS-neutralizing proteins, the enzymes of polyamine metabolisms and proline and urea cycles, as well as calcium ion transporters between cell lines, are shown. The data obtained are important for understanding the redox biology of viral hepatitis and elucidating the influence of the laboratory models used.
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Affiliation(s)
- Olga N. Ivanova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - George S. Krasnov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Anastasiya V. Snezhkina
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Anna V. Kudryavtseva
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Vyacheslav S. Fedorov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Natalia F. Zakirova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Michail V. Golikov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Sergey N. Kochetkov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Birke Bartosch
- Lyon Cancer Research Center, Université Claude Bernard Lyon 1, INSERM U1052, CNRS 5286, 69008 Lyon, France
| | | | - Alexander V. Ivanov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
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18
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Müller FA, Stamou M, Englert FH, Frenzel O, Diedrich S, Suter-Dick L, Wambaugh JF, Sturla SJ. In vitro to in vivo extrapolation and high-content imaging for simultaneous characterization of chemically induced liver steatosis and markers of hepatotoxicity. Arch Toxicol 2023; 97:1701-1721. [PMID: 37046073 PMCID: PMC10182956 DOI: 10.1007/s00204-023-03490-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/21/2023] [Accepted: 03/21/2023] [Indexed: 04/14/2023]
Abstract
Chemically induced steatosis is characterized by lipid accumulation associated with mitochondrial dysfunction, oxidative stress and nucleus distortion. New approach methods integrating in vitro and in silico models are needed to identify chemicals that may induce these cellular events as potential risk factors for steatosis and associated hepatotoxicity. In this study we used high-content imaging for the simultaneous quantification of four cellular markers as sentinels for hepatotoxicity and steatosis in chemically exposed human liver cells in vitro. Furthermore, we evaluated the results with a computational model for the extrapolation of human oral equivalent doses (OED). First, we tested 16 reference chemicals with known capacities to induce cellular alterations in nuclear morphology, lipid accumulation, mitochondrial membrane potential and oxidative stress. Then, using physiologically based pharmacokinetic modeling and reverse dosimetry, OEDs were extrapolated from data of any stimulated individual sentinel response. The extrapolated OEDs were confirmed to be within biologically relevant exposure ranges for the reference chemicals. Next, we tested 14 chemicals found in food, selected from thousands of putative chemicals on the basis of structure-based prediction for nuclear receptor activation. Amongst these, orotic acid had an extrapolated OED overlapping with realistic exposure ranges. Thus, we were able to characterize known steatosis-inducing chemicals as well as data-scarce food-related chemicals, amongst which we confirmed orotic acid to induce hepatotoxicity. This strategy addresses needs of next generation risk assessment and can be used as a first chemical prioritization hazard screening step in a tiered approach to identify chemical risk factors for steatosis and hepatotoxicity-associated events.
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Affiliation(s)
- Fabrice A Müller
- Department of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 9, 8092, Zurich, Switzerland
| | - Marianna Stamou
- Department of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 9, 8092, Zurich, Switzerland
| | - Felix H Englert
- Department of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 9, 8092, Zurich, Switzerland
| | - Ole Frenzel
- Department of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 9, 8092, Zurich, Switzerland
| | - Sabine Diedrich
- Department of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 9, 8092, Zurich, Switzerland
| | - Laura Suter-Dick
- School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, 4132, Muttenz, Switzerland
- Swiss Centre for Applied Human Toxicology (SCAHT), 4001, Basel, Switzerland
| | - John F Wambaugh
- Center for Computational Toxicology and Exposure, Office of Research and Development, United States Environmental Protection Agency, Research Triangle Park, Durham, NC, 27711, USA
| | - Shana J Sturla
- Department of Health Sciences and Technology, ETH Zurich, Schmelzbergstrasse 9, 8092, Zurich, Switzerland.
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19
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Ma Y, Hu L, Tang J, Guo W, Feng Y, Liu Y, Tang F. Three-Dimensional Cell Co-Culture Liver Models and Their Applications in Pharmaceutical Research. Int J Mol Sci 2023; 24:ijms24076248. [PMID: 37047220 PMCID: PMC10094553 DOI: 10.3390/ijms24076248] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Revised: 03/14/2023] [Accepted: 03/22/2023] [Indexed: 03/29/2023] Open
Abstract
As the primary site for the biotransformation of drugs, the liver is the most focused on organ type in pharmaceutical research. However, despite being widely used in pharmaceutical research, animal models have inherent species differences, while two-dimensional (2D) liver cell monocultures or co-cultures and three-dimensional (3D) liver cell monoculture in vitro liver models do not sufficiently represent the complexity of the human liver’s structure and function, making the evaluation results from these tools less reliable. Therefore, there is a pressing need to develop more representative in vitro liver models for pharmaceutical research. Fortunately, an exciting new development in recent years has been the emergence of 3D liver cell co-culture models. These models hold great promise as in vitro pharmaceutical research tools, because they can reproduce liver structure and function more practically. This review begins by explaining the structure and main cell composition of the liver, before introducing the potential advantages of 3D cell co-culture liver models for pharmaceutical research. We also discuss the main sources of hepatocytes and the 3D cell co-culture methods used in constructing these models. In addition, we explore the applications of 3D cell co-culture liver models with different functional states and suggest prospects for their further development.
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20
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Diprospero TJ, Brown LG, Fachko TD, Lockett MR. HepaRG cells undergo increased levels of post-differentiation patterning in physiologic conditions when maintained as 3D cultures in paper-based scaffolds. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2023:2023.01.16.524330. [PMID: 36711996 PMCID: PMC9882149 DOI: 10.1101/2023.01.16.524330] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/21/2023]
Abstract
Monolayer cultures of hepatocytes lack many aspects of the liver sinusoid, including a tissue-level organization that results from extracellular matrix interactions and gradients of soluble molecules that span from the portal triad to the central vein. We measured the activity and transcript levels of drug-metabolizing enzymes in HepaRG cells maintained in three different culture configurations: as monolayers, seeded onto paper scaffolds that were pre-loaded with a collagen matrix, and when seeded directly into the paper scaffolds as a cell-laden gel. Drug metabolism was significantly decreased in the presence of the paper scaffolds compared to monolayer configurations when cells were exposed to standard culture conditions. Despite this decreased function, transcript levels suggest the cells undergo increased polarization and adopt a biliary-like character in the paper scaffolds, including the increased expression of transporter proteins (e.g., ABCB11 and SLOC1B1) and the KRT19 cholangiocyte marker. When exposed to representative periportal or perivenous culture conditions, we observed in vivo zonal-like patterns, including increased cytochrome P450 (CYP) activity and transcript levels in the perivenous condition. This increased CYP activity is more pronounced in the laden configuration, supporting the need to include multiple aspects of the liver microenvironment to observe the post-differentiation processing of hepatocytes.
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Affiliation(s)
- Thomas J. Diprospero
- Department of Chemistry, University of North Carolina at Chapel Hill, Kenan and Caudill Laboratories, Chapel Hill, NC 27599-3290, United States
| | - Lauren G. Brown
- Department of Chemistry, University of North Carolina at Chapel Hill, Kenan and Caudill Laboratories, Chapel Hill, NC 27599-3290, United States
| | - Trevor D. Fachko
- Department of Chemistry, University of North Carolina at Chapel Hill, Kenan and Caudill Laboratories, Chapel Hill, NC 27599-3290, United States
| | - Matthew R. Lockett
- Department of Chemistry, University of North Carolina at Chapel Hill, Kenan and Caudill Laboratories, Chapel Hill, NC 27599-3290, United States
- Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 37599-7295, United States
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21
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Smirnova OA, Ivanova ON, Fedyakina IT, Yusubalieva GM, Baklaushev VP, Yanvarev DV, Kechko OI, Mitkevich VA, Vorobyev PO, Fedorov VS, Bartosch B, Valuev-Elliston VT, Lipatova AL, Ivanov AV. SARS-CoV-2 Establishes a Productive Infection in Hepatoma and Glioblastoma Multiforme Cell Lines. Cancers (Basel) 2023; 15:632. [PMID: 36765590 PMCID: PMC9913867 DOI: 10.3390/cancers15030632] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/18/2022] [Revised: 01/09/2023] [Accepted: 01/18/2023] [Indexed: 01/20/2023] Open
Abstract
Severe acute respiratory syndrome associated coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and rapidly caused a pandemic that led to the death of >6 million people due to hypercoagulation and cytokine storm. In addition, SARS-CoV-2 triggers a wide array of pathologies, including liver dysfunction and neurological disorders. It remains unclear if these events are due to direct infection of the respective tissues or result from systemic inflammation. Here, we explored the possible infection of hepatic and CNS cell lines by SARS-CoV-2. We show that even moderate expression levels of the angiotensin-converting enzyme 2 (ACE2) are sufficient for productive infection. SARS-CoV-2 infects hepatoma Huh7.5 and HepG2 cells but not non-transformed liver progenitor or hepatocyte/cholangiocyte-like HepaRG cells. However, exposure to the virus causes partial dedifferentiation of HepaRG cells. SARS-CoV-2 can also establish efficient replication in some low-passage, high-grade glioblastoma cell lines. In contrast, embryonal primary astrocytes or neuroblastoma cells did not support replication of the virus. Glioblastoma cell permissiveness is associated with defects in interferon production. Overall, these results suggest that liver dysfunction during COVID-19 is not due to infection of these tissues by SARS-CoV-2. Furthermore, tumors may potentially serve as reservoirs for the virus during infection.
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Affiliation(s)
- Olga A. Smirnova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Olga N. Ivanova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Irina T. Fedyakina
- Gamaleya National Research Centre for Epidemiology and Microbiology of the Ministry of Russia, 123098 Moscow, Russia
| | - Gaukhar M. Yusubalieva
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
- Federal Research and Clinical Center of Specialized Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia, 115682 Moscow, Russia
| | - Vladimir P. Baklaushev
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
- Federal Research and Clinical Center of Specialized Medical Care and Medical Technologies of the Federal Medical and Biological Agency of Russia, 115682 Moscow, Russia
| | - Dmitry V. Yanvarev
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Olga I. Kechko
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Vladimir A. Mitkevich
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Pavel O. Vorobyev
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Vyacheslav S. Fedorov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Birke Bartosch
- Lyon Cancer Research Center/INSERM U1052, 69008 Lyon, France
| | | | - Anastasiya L. Lipatova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
| | - Alexander V. Ivanov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia
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22
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Bellanti F, Mangieri D, di Bello G, Lo Buglio A, Pannone G, Pedicillo MC, Fersini A, Dobrakowski M, Kasperczyk A, Kasperczyk S, Vendemiale G. Redox-Dependent Modulation of Human Liver Progenitor Cell Line Fate. Int J Mol Sci 2023; 24:1934. [PMID: 36768260 PMCID: PMC9916526 DOI: 10.3390/ijms24031934] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2023] [Revised: 01/13/2023] [Accepted: 01/16/2023] [Indexed: 01/20/2023] Open
Abstract
Redox homeostasis is determinant in the modulation of quiescence/self-renewal/differentiation of stem cell lines. The aim of this study consisted of defining the impact of redox modifications on cell fate in a human hepatic progenitor line. To achieve this, the HepaRG cell line, which shows oval ductular bipotent characteristics, was used. The impact of redox status on the balance between self-renewal and differentiation of HepaRG cells was investigated using different methodological approaches. A bioinformatic analysis initially proved that the trans-differentiation of HepaRG toward bipotent progenitors is associated with changes in redox metabolism. We then exposed confluent HepaRG (intermediate differentiation phase) to oxidized (H2O2) or reduced (N-acetylcysteine) extracellular environments, observing that oxidation promotes the acquisition of a mature HepaRG phenotype, while a reduced culture medium stimulates de-differentiation. These results were finally confirmed through pharmacological modulation of the nuclear factor (erythroid-derived 2)-like 2 (NRF2), a principal modulator of the antioxidant response, in confluent HepaRG. NRF2 inhibition led to intracellular pro-oxidative status and HepaRG differentiation, while its activation was associated with low levels of reactive species and de-differentiation. In conclusion, this study shows that both intra- and extracellular redox balance are crucial in the determination of HepaRG fate. The impact of redox status in the differentiation potential of HepaRG cells is significant on the utilization of this cell line in pre-clinical studies.
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Affiliation(s)
- Francesco Bellanti
- Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy
| | - Domenica Mangieri
- Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy
| | - Giorgia di Bello
- Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy
| | - Aurelio Lo Buglio
- Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy
| | - Giuseppe Pannone
- Department of Clinical and Experimental Medicine, University of Foggia, 71122 Foggia, Italy
| | | | - Alberto Fersini
- Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy
| | - Michał Dobrakowski
- Department of Biochemistry, Faculty of Medical Sciences in Zabrze, Medical University of Silesia, 41-800 Katowice, Poland
| | - Aleksandra Kasperczyk
- Department of Biochemistry, Faculty of Medical Sciences in Zabrze, Medical University of Silesia, 41-800 Katowice, Poland
| | - Sławomir Kasperczyk
- Department of Biochemistry, Faculty of Medical Sciences in Zabrze, Medical University of Silesia, 41-800 Katowice, Poland
| | - Gianluigi Vendemiale
- Department of Medical and Surgical Sciences, University of Foggia, 71122 Foggia, Italy
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23
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Diprospero TJ, Brown LG, Fachko TD, Lockett MR. HepaRG cells undergo increased levels of post-differentiation patterning in physiologic conditions when maintained as 3D cultures in paper-based scaffolds. RESEARCH SQUARE 2023:rs.3.rs-2473387. [PMID: 36711963 PMCID: PMC9882668 DOI: 10.21203/rs.3.rs-2473387/v1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Indexed: 01/20/2023]
Abstract
Monolayer cultures of hepatocytes lack many aspects of the liver sinusoid, including a tissue-level organization that results from extracellular matrix interactions and gradients of soluble molecules that span from the portal triad to the central vein. We measured the activity and transcript levels of drug-metabolizing enzymes in HepaRG cells maintained in three different culture configurations: as monolayers, seeded onto paper scaffolds that were pre-loaded with a collagen matrix, and when seeded directly into the paper scaffolds as a cell-laden gel. Drug metabolism was significantly decreased in the presence of the paper scaffolds compared to monolayer configurations when cells were exposed to standard culture conditions. Despite this decreased function, transcript levels suggest the cells undergo increased polarization and adopt a biliary-like character in the paper scaffolds, including the increased expression of transporter proteins (e.g., ABCB11 and SLOC1B1) and the KRT19 cholangiocyte marker. When exposed to representative periportal or perivenous culture conditions, we observed in vivo zonal-like patterns, including increased cytochrome P450 (CYP) activity and transcript levels in the perivenous condition. This increased CYP activity is more pronounced in the laden configuration, supporting the need to include multiple aspects of the liver microenvironment to observe the post-differentiation processing of hepatocytes.
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24
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Chen G, Chang Z, Yuan P, Wang S, Yang Y, Liang X, Zhao D. Late-stage functionalization of 5-nitrofurans derivatives and their antibacterial activities. RSC Adv 2023; 13:3204-3209. [PMID: 36756397 PMCID: PMC9853512 DOI: 10.1039/d2ra07676d] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2022] [Accepted: 01/16/2023] [Indexed: 01/21/2023] Open
Abstract
Structure modification of drugs is a reliable way to optimize lead compounds, among which the most striking and direct method is late-stage functionalization (LSF). Here, we employed the Cu-catalyzed C-H LSF to modify 5-nitrofuran drugs. A series of modifications have been carried out including hydroxylation, methylation, azidination, cyanation, arylation, etc. Antibacterial activities of all compounds in vitro were measured. The results showed that compound 1 and compound 18 were the most active among all compounds. Meanwhile, the cell cytotoxicity assays of potent compounds 1, 3, 4, 5 & 18 and the parent drug FZD were conducted.
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Affiliation(s)
- Geshuyi Chen
- The First Clinical Medical College, Lanzhou University Lanzhou China
| | - Zhe Chang
- Guangdong Provincial Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University Guangzhou China
| | - Pei Yuan
- The First Clinical Medical College, Lanzhou University Lanzhou China
| | - Si Wang
- Guangdong Provincial Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University Guangzhou China
| | - Yongxiu Yang
- The First Clinical Medical College, Lanzhou University Lanzhou China .,The First Clinical Medical College, Lanzhou University. Department of Obstetrics and Gynecology, The First Hospital of Lanzhou University, Key Laboratory for Gynecologic Oncology Lanzhou 730000 Gansu Province China .,Lead Contact China
| | - Xiaolei Liang
- The First Clinical Medical College, Lanzhou University Lanzhou China .,The First Clinical Medical College, Lanzhou University. Department of Obstetrics and Gynecology, The First Hospital of Lanzhou University, Key Laboratory for Gynecologic Oncology Lanzhou 730000 Gansu Province China
| | - Depeng Zhao
- Guangdong Provincial Key Laboratory of Chiral Molecule and Drug Discovery, School of Pharmaceutical Sciences, Sun Yat-Sen University Guangzhou China
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25
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Barnault R, Verzeroli C, Fournier C, Michelet M, Redavid AR, Chicherova I, Plissonnier ML, Adrait A, Khomich O, Chapus F, Richaud M, Hervieu M, Reiterer V, Centonze FG, Lucifora J, Bartosch B, Rivoire M, Farhan H, Couté Y, Mirakaj V, Decaens T, Mehlen P, Gibert B, Zoulim F, Parent R. Hepatic inflammation elicits production of proinflammatory netrin-1 through exclusive activation of translation. Hepatology 2022; 76:1345-1359. [PMID: 35253915 DOI: 10.1002/hep.32446] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/02/2021] [Revised: 02/16/2022] [Accepted: 02/17/2022] [Indexed: 12/18/2022]
Abstract
BACKGROUND AND AIMS Netrin-1 displays protumoral properties, though the pathological contexts and processes involved in its induction remain understudied. The liver is a major model of inflammation-associated cancer development, leading to HCC. APPROACH AND RESULTS A panel of cell biology and biochemistry approaches (reverse transcription quantitative polymerase chain reaction, reporter assays, run-on, polysome fractionation, cross linking immunoprecipitation, filter binding assay, subcellular fractionation, western blotting, immunoprecipitation, stable isotope labeling by amino acids in cell culture) on in vitro-grown primary hepatocytes, human liver cell lines, mouse samples and clinical samples was used. We identify netrin-1 as a hepatic inflammation-inducible factor and decipher its mode of activation through an exhaustive eliminative approach. We show that netrin-1 up-regulation relies on a hitherto unknown mode of induction, namely its exclusive translational activation. This process includes the transfer of NTN1 (netrin-1) mRNA to the endoplasmic reticulum and the direct interaction between the Staufen-1 protein and this transcript as well as netrin-1 mobilization from its cell-bound form. Finally, we explore the impact of a phase 2 clinical trial-tested humanized anti-netrin-1 antibody (NP137) in two distinct, toll-like receptor (TLR) 2/TLR3/TLR6-dependent, hepatic inflammatory mouse settings. We observe a clear anti-inflammatory activity indicating the proinflammatory impact of netrin-1 on several chemokines and Ly6C+ macrophages. CONCLUSIONS These results identify netrin-1 as an inflammation-inducible factor in the liver through an atypical mechanism as well as its contribution to hepatic inflammation.
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Affiliation(s)
- Romain Barnault
- Pathogenesis of Chronic Hepatitis B and C Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France.,University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France
| | - Claire Verzeroli
- Pathogenesis of Chronic Hepatitis B and C Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France.,University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France
| | - Carole Fournier
- Institute for Advanced Biosciences, Inserm U1209, University of Grenoble-Alpes, La Tronche, France
| | - Maud Michelet
- Pathogenesis of Chronic Hepatitis B and C Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France.,University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France
| | - Anna Rita Redavid
- University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France.,Apoptosis, Cancer and Development Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France
| | - Ievgeniia Chicherova
- Pathogenesis of Chronic Hepatitis B and C Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France.,University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France
| | - Marie-Laure Plissonnier
- University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France.,Inserm U1052, Cancer Research Centre of Lyon, Lyon, France
| | - Annie Adrait
- University of Grenoble-Alpes, Inserm, CEA, UMR BioSanté U1292, CNRS CEA FR2048, Grenoble, France
| | - Olga Khomich
- Pathogenesis of Chronic Hepatitis B and C Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France.,University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France
| | - Fleur Chapus
- Single Cell Dynamics Group, Epigenetics and Stem Cell Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, Durham, North Carolina, USA
| | - Mathieu Richaud
- University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France.,Apoptosis, Cancer and Development Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France
| | - Maëva Hervieu
- University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France.,Apoptosis, Cancer and Development Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France
| | - Veronika Reiterer
- Institute of Pathophysiology, Medical University of Innsbruck, Innsbruck, Austria
| | | | - Julie Lucifora
- Pathogenesis of Chronic Hepatitis B and C Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France.,University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France
| | - Birke Bartosch
- Pathogenesis of Chronic Hepatitis B and C Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France.,University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France
| | - Michel Rivoire
- Léon Bérard Cancer Center, Lyon, France.,Université Lyon 1, Lyon, France
| | - Hesso Farhan
- Institute of Pathophysiology, Medical University of Innsbruck, Innsbruck, Austria
| | - Yohann Couté
- University of Grenoble-Alpes, Inserm, CEA, UMR BioSanté U1292, CNRS CEA FR2048, Grenoble, France
| | - Valbona Mirakaj
- Department of Anesthesiology and Intensive Care Medicine, University Hospital of Tuebingen, Eberhard-Karls University, Tuebingen, Germany
| | - Thomas Decaens
- Institute for Advanced Biosciences, Inserm U1209, University of Grenoble-Alpes, La Tronche, France
| | - Patrick Mehlen
- University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France.,Apoptosis, Cancer and Development Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France
| | - Benjamin Gibert
- University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France.,Apoptosis, Cancer and Development Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France
| | - Fabien Zoulim
- Pathogenesis of Chronic Hepatitis B and C Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France.,University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France.,Service of Hepato-Gastroenterology, Hospices Civils de Lyon, Lyon, France
| | - Romain Parent
- Pathogenesis of Chronic Hepatitis B and C Laboratory - LabEx DEVweCAN, Inserm U1052, Cancer Research Centre of Lyon, Lyon, France.,University of Lyon, Lyon, France.,University Lyon 1, Institut des Sciences Pharmaceutiques et Biologiques, Lyon, France.,CNRS UMR5286, Lyon, France.,Centre Léon Bérard, Lyon, France
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26
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Dolicka D, Zahoran S, Correia de Sousa M, Gjorgjieva M, Sempoux C, Fournier M, Maeder C, Collart MA, Foti M, Sobolewski C. TIA1 Loss Exacerbates Fatty Liver Disease but Exerts a Dual Role in Hepatocarcinogenesis. Cancers (Basel) 2022; 14:cancers14071704. [PMID: 35406476 PMCID: PMC8997004 DOI: 10.3390/cancers14071704] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2022] [Revised: 03/24/2022] [Accepted: 03/25/2022] [Indexed: 11/16/2022] Open
Abstract
Alterations in specific RNA-binding protein expression/activity importantly contribute to the development of fatty liver disease (FLD) and hepatocellular carcinoma (HCC). In particular, adenylate–uridylate-rich element binding proteins (AUBPs) were reported to control the post-transcriptional regulation of genes involved in both metabolic and cancerous processes. Herein, we investigated the pathophysiological functions of the AUBP, T-cell-restricted intracellular antigen-1 (TIA1) in the development of FLD and HCC. Analysis of TIA1 expression in mouse and human models of FLD and HCC indicated that TIA1 is downregulated in human HCC. In vivo silencing of TIA1 using AAV8-delivered shRNAs in mice worsens hepatic steatosis and fibrosis induced by a methionine and choline-deficient diet and increases the hepatic tumor burden in liver-specific PTEN knockout (LPTENKO) mice. In contrast, our in vitro data indicated that TIA1 expression promoted proliferation and migration in HCC cell lines, thus suggesting a dual and context-dependent role for TIA1 in tumor initiation versus progression. Consistent with a dual function of TIA1 in tumorigenesis, translatome analysis revealed that TIA1 appears to control the expression of both pro- and anti-tumorigenic factors in hepatic cancer cells. This duality of TIA1′s function in hepatocarcinogenesis calls for cautiousness when considering TIA1 as a therapeutic target or biomarker in HCC.
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Affiliation(s)
- Dobrochna Dolicka
- Department of Cell Physiology and Metabolism, Translational Research Centre in Onco-Hematology (CRTOH), Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland; (D.D.); (M.C.d.S.); (M.G.); (M.F.); (C.M.); (M.F.)
| | - Szabolcs Zahoran
- Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland; (S.Z.); (M.A.C.)
| | - Marta Correia de Sousa
- Department of Cell Physiology and Metabolism, Translational Research Centre in Onco-Hematology (CRTOH), Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland; (D.D.); (M.C.d.S.); (M.G.); (M.F.); (C.M.); (M.F.)
| | - Monika Gjorgjieva
- Department of Cell Physiology and Metabolism, Translational Research Centre in Onco-Hematology (CRTOH), Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland; (D.D.); (M.C.d.S.); (M.G.); (M.F.); (C.M.); (M.F.)
| | - Christine Sempoux
- Service of Clinical Pathology, Institute of Pathology, Lausanne University Hospital and University of Lausanne, CH-1007 Lausanne, Switzerland;
| | - Margot Fournier
- Department of Cell Physiology and Metabolism, Translational Research Centre in Onco-Hematology (CRTOH), Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland; (D.D.); (M.C.d.S.); (M.G.); (M.F.); (C.M.); (M.F.)
| | - Christine Maeder
- Department of Cell Physiology and Metabolism, Translational Research Centre in Onco-Hematology (CRTOH), Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland; (D.D.); (M.C.d.S.); (M.G.); (M.F.); (C.M.); (M.F.)
| | - Martine A. Collart
- Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland; (S.Z.); (M.A.C.)
| | - Michelangelo Foti
- Department of Cell Physiology and Metabolism, Translational Research Centre in Onco-Hematology (CRTOH), Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland; (D.D.); (M.C.d.S.); (M.G.); (M.F.); (C.M.); (M.F.)
| | - Cyril Sobolewski
- Department of Cell Physiology and Metabolism, Translational Research Centre in Onco-Hematology (CRTOH), Faculty of Medicine, University of Geneva, CH-1211 Geneva, Switzerland; (D.D.); (M.C.d.S.); (M.G.); (M.F.); (C.M.); (M.F.)
- Correspondence: or
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27
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Stanley LA, Wolf CR. Through a glass, darkly? HepaRG and HepG2 cells as models of human phase I drug metabolism. Drug Metab Rev 2022; 54:46-62. [PMID: 35188018 DOI: 10.1080/03602532.2022.2039688] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Abstract
The pharmacokinetic and safety assessment of drug candidates is becoming increasingly dependent upon in vitro models of hepatic metabolism and toxicity. Predominant among these is the HepG2 cell line, although HepaRG is becoming increasingly popular because of its perceived closer resemblance to human hepatocytes. We review the functionality of these cell lines in terms of Phase I protein expression, basal cytochrome P450-dependent activity, and utility in P450 induction studies. Our analysis indicates that HepG2 cells are severely compromised: proteomic studies show that they express few key proteins in common with hepatocytes and they lack drug-metabolizing capacity. Differentiated HepaRGs are more hepatocyte-like than HepG2s, but they also have limitations, and it is difficult to assess their utility because of the enormous variability in data reported, possibly arising from the complex differentiation protocols required to obtain hepatocyte-like cells. This is exacerbated by the use of DMSO in the induction protocol, together with proprietary supplements whose composition is a commercial secret. We conclude that, while currently available data on the utility of HepaRG generates a confusing picture, this line does have potential utility in drug metabolism studies. However, to allow studies to be compared directly a standardized, reproducible differentiation protocol is essential and the cell line's functionality in terms of known mechanisms of P450 regulation must be demonstrated. We, therefore, support the development of regulatory guidelines for the use of HepaRGs in induction studies as a first step in generating a database of consistent, reliable data.
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Affiliation(s)
- Lesley A Stanley
- Consultant in Investigative Toxicology, Linlithgow, UK.,School of Applied Sciences, Edinburgh Napier University, Edinburgh, UK
| | - C Roland Wolf
- Systems Medicine, School of Medicine, University of Dundee, Jacqui Wood Cancer Centre, Ninewells Hospital, Dundee, UK
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28
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Youhanna S, Kemas AM, Preiss L, Zhou Y, Shen JX, Cakal SD, Paqualini FS, Goparaju SK, Shafagh RZ, Lind JU, Sellgren CM, Lauschke VM. Organotypic and Microphysiological Human Tissue Models for Drug Discovery and Development-Current State-of-the-Art and Future Perspectives. Pharmacol Rev 2022; 74:141-206. [PMID: 35017176 DOI: 10.1124/pharmrev.120.000238] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2020] [Accepted: 10/12/2021] [Indexed: 12/11/2022] Open
Abstract
The number of successful drug development projects has been stagnant for decades despite major breakthroughs in chemistry, molecular biology, and genetics. Unreliable target identification and poor translatability of preclinical models have been identified as major causes of failure. To improve predictions of clinical efficacy and safety, interest has shifted to three-dimensional culture methods in which human cells can retain many physiologically and functionally relevant phenotypes for extended periods of time. Here, we review the state of the art of available organotypic culture techniques and critically review emerging models of human tissues with key importance for pharmacokinetics, pharmacodynamics, and toxicity. In addition, developments in bioprinting and microfluidic multiorgan cultures to emulate systemic drug disposition are summarized. We close by highlighting important trends regarding the fabrication of organotypic culture platforms and the choice of platform material to limit drug absorption and polymer leaching while supporting the phenotypic maintenance of cultured cells and allowing for scalable device fabrication. We conclude that organotypic and microphysiological human tissue models constitute promising systems to promote drug discovery and development by facilitating drug target identification and improving the preclinical evaluation of drug toxicity and pharmacokinetics. There is, however, a critical need for further validation, benchmarking, and consolidation efforts ideally conducted in intersectoral multicenter settings to accelerate acceptance of these novel models as reliable tools for translational pharmacology and toxicology. SIGNIFICANCE STATEMENT: Organotypic and microphysiological culture of human cells has emerged as a promising tool for preclinical drug discovery and development that might be able to narrow the translation gap. This review discusses recent technological and methodological advancements and the use of these systems for hit discovery and the evaluation of toxicity, clearance, and absorption of lead compounds.
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Affiliation(s)
- Sonia Youhanna
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Aurino M Kemas
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Lena Preiss
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Yitian Zhou
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Joanne X Shen
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Selgin D Cakal
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Francesco S Paqualini
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Sravan K Goparaju
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Reza Zandi Shafagh
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Johan Ulrik Lind
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Carl M Sellgren
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
| | - Volker M Lauschke
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden (S.Y., A.M.K., L.P., Y.Z., J.X.S., S.K.G., R.Z.S., C.M.S., V.M.L.); Department of Drug Metabolism and Pharmacokinetics (DMPK), Merck KGaA, Darmstadt, Germany (L.P.); Department of Health Technology, Technical University of Denmark, Lyngby, Denmark (S.D.C., J.U.L.); Synthetic Physiology Laboratory, Department of Civil Engineering and Architecture, University of Pavia, Pavia, Italy (F.S.P.); Division of Micro- and Nanosystems, KTH Royal Institute of Technology, Stockholm, Sweden (Z.S.); and Dr Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany (V.M.L.)
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Desoteux M, Louis C, Bévant K, Glaise D, Coulouarn C. A Minimal Subset of Seven Genes Associated with Tumor Hepatocyte Differentiation Predicts a Poor Prognosis in Human Hepatocellular Carcinoma. Cancers (Basel) 2021; 13:5624. [PMID: 34830779 PMCID: PMC8616205 DOI: 10.3390/cancers13225624] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/01/2021] [Revised: 11/03/2021] [Accepted: 11/05/2021] [Indexed: 12/23/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is a deadly cancer worldwide as a result of a frequent late diagnosis which limits the therapeutic options. Tumor progression in HCC is closely correlated with the dedifferentiation of hepatocytes, the main parenchymal cells in the liver. Here, we hypothesized that the expression level of genes reflecting the differentiation status of tumor hepatocytes could be clinically relevant in defining subsets of patients with different clinical outcomes. To test this hypothesis, an integrative transcriptomics approach was used to stratify a cohort of 139 HCC patients based on a gene expression signature established in vitro in the HepaRG cell line using well-controlled culture conditions recapitulating tumor hepatocyte differentiation. The HepaRG model was first validated by identifying a robust gene expression signature associated with hepatocyte differentiation and liver metabolism. In addition, the signature was able to distinguish specific developmental stages in mice. More importantly, the signature identified a subset of human HCC associated with a poor prognosis and cancer stem cell features. By using an independent HCC dataset (TCGA consortium), a minimal subset of seven differentiation-related genes was shown to predict a reduced overall survival, not only in patients with HCC but also in other types of cancers (e.g., kidney, pancreas, skin). In conclusion, the study identified a minimal subset of seven genes reflecting the differentiation status of tumor hepatocytes and clinically relevant for predicting the prognosis of HCC patients.
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Affiliation(s)
- Matthis Desoteux
- Inserm, Univ. Rennes, UMR1242, Chemistry Oncogenesis Stress Signaling (COSS), 35042 Rennes, France; (M.D.); (C.L.); (K.B.)
| | - Corentin Louis
- Inserm, Univ. Rennes, UMR1242, Chemistry Oncogenesis Stress Signaling (COSS), 35042 Rennes, France; (M.D.); (C.L.); (K.B.)
| | - Kevin Bévant
- Inserm, Univ. Rennes, UMR1242, Chemistry Oncogenesis Stress Signaling (COSS), 35042 Rennes, France; (M.D.); (C.L.); (K.B.)
| | - Denise Glaise
- Inserm, Univ. Rennes, UMR991, Liver Metabolisms and Cancer, 35043 Rennes, France;
| | - Cédric Coulouarn
- Inserm, Univ. Rennes, UMR1242, Chemistry Oncogenesis Stress Signaling (COSS), 35042 Rennes, France; (M.D.); (C.L.); (K.B.)
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30
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Zhao Z, Wang Y, Wu Y, Li D, Zhang T, Ma Y, Teng X, Zuo W. Single-cell analysis defines the lineage plasticity of stem cells in cervix epithelium. CELL REGENERATION 2021; 10:36. [PMID: 34719766 PMCID: PMC8558147 DOI: 10.1186/s13619-021-00096-2] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/15/2021] [Accepted: 09/22/2021] [Indexed: 11/10/2022]
Abstract
Information about the dynamic change and post-injury regeneration of cervical epithelium is relatively rare, even though it is tightly related to gynecologic malignancy. Here, using a feeder cell-based culturing system, we stably cloned mouse and human P63 and KRT5 expressing cells from the adult cervix as putative cervical stem/progenitor cells (CVSCs). When subjected to differentiation, the cultured cells gave rise to mature cervical epithelium by differentiating into squamous or glandular cells. The ability of endogenous mouse CVSCs to reconstitute cervical epithelium after injury was also evident from the genetic lineage tracing experiments. Single-cell transcriptomic analysis further classified the CVSCs into three subtypes and delineated their bi-lineage differentiation roadmap by pseudo-time analysis. We also tracked the real-time differentiation routes of two representing single CVSC lines in vitro and found that they recapitulated the predicted roadmap in pseudo-time analysis. Signaling pathways including Wnt, TGF-beta, Notch and EGFR were found to regulate the cervical epithelial hierarchy and implicated the different roles of distinct types of cells in tissue homeostasis and tumorigenesis. Collectively, the above data provide a cloning system to achieve stable in vitro culture of a bi-lineage stem/progenitor cell population in the cervix, which has profound implications for our understanding of the cervix stem/progenitor cell function in homeostasis, regeneration, and disease and could be helpful for developing stem cell-based therapies in future.
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Affiliation(s)
- Zixian Zhao
- East Hospital, School of Medicine, Tongji University, Shanghai, China
| | - Yujia Wang
- East Hospital, School of Medicine, Tongji University, Shanghai, China.,Super Organ R&D Center, Regend Therapeutics, Shanghai, China
| | - Yingchuan Wu
- East Hospital, School of Medicine, Tongji University, Shanghai, China.,Shanghai Jincai East Secondary School, Shanghai, China
| | - Dandan Li
- East Hospital, School of Medicine, Tongji University, Shanghai, China
| | - Ting Zhang
- Super Organ R&D Center, Regend Therapeutics, Shanghai, China
| | - Yu Ma
- Super Organ R&D Center, Regend Therapeutics, Shanghai, China
| | - Xiaoming Teng
- Shanghai First Maternity and Infant Hospital, Tongji University, Shanghai, China
| | - Wei Zuo
- East Hospital, School of Medicine, Tongji University, Shanghai, China. .,Super Organ R&D Center, Regend Therapeutics, Shanghai, China. .,Ningxia Medical University, Yinchuan, China. .,The First Affiliated Hospital, Guangzhou Medical University, Guangzhou, China.
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31
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Bellanti F, di Bello G, Tamborra R, Amatruda M, Lo Buglio A, Dobrakowski M, Kasperczyk A, Kasperczyk S, Serviddio G, Vendemiale G. Impact of senescence on the transdifferentiation process of human hepatic progenitor-like cells. World J Stem Cells 2021; 13:1595-1609. [PMID: 34786160 PMCID: PMC8567448 DOI: 10.4252/wjsc.v13.i10.1595] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 04/26/2021] [Revised: 06/14/2021] [Accepted: 08/23/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Senescence is characterized by a decline in hepatocyte function, with impairment of metabolism and regenerative capacity. Several models that duplicate liver functions in vitro are essential tools for studying drug metabolism, liver diseases, and organ regeneration. The human HepaRG cell line represents an effective model for the study of liver metabolism and hepatic progenitors. However, the impact of senescence on HepaRG cells is not yet known. AIM To characterize the effects of senescence on the transdifferentiation capacity and mitochondrial metabolism of human HepaRG cells. METHODS We compared the transdifferentiation capacity of cells over 10 (passage 10 [P10]) vs P20. Aging was evaluated by senescence-associated (SA) beta-galactosidase activity and the comet assay. HepaRG transdifferentiation was analyzed by confocal microscopy and flow cytometry (expression of cluster of differentiation 49a [CD49a], CD49f, CD184, epithelial cell adhesion molecule [EpCAM], and cytokeratin 19 [CK19]), quantitative PCR analysis (expression of albumin, cytochrome P450 3A4 [CYP3A4], γ-glutamyl transpeptidase [γ-GT], and carcinoembryonic antigen [CEA]), and functional analyses (albumin secretion, CYP3A4, and γ-GT). Mitochondrial respiration and the ATP and nicotinamide adenine dinucleotide (NAD+)/NAD with hydrogen (NADH) content were also measured. RESULTS SA β-galactosidase staining was higher in P20 than P10 HepaRG cells; in parallel, the comet assay showed consistent DNA damage in P20 HepaRG cells. With respect to P10, P20 HepaRG cells exhibited a reduction of CD49a, CD49f, CD184, EpCAM, and CK19 after the induction of transdifferentiation. Furthermore, lower gene expression of albumin, CYP3A4, and γ-GT, as well as reduced albumin secretion capacity, CYP3A4, and γ-GT activity were reported in transdifferentiated P20 compared to P10 cells. By contrast, the gene expression level of CEA was not reduced by transdifferentiation in P20 cells. Of note, both cellular and mitochondrial oxygen consumption was lower in P20 than in P10 transdifferentiated cells. Finally, both ATP and NAD+/NADH were depleted in P20 cells with respect to P10 cells. CONCLUSION SA mitochondrial dysfunction may limit the transdifferentiation potential of HepaRG cells, with consequent impairment of metabolic and regenerative properties, which may alter applications in basic studies.
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Affiliation(s)
- Francesco Bellanti
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy.
| | - Giorgia di Bello
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Rosanna Tamborra
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Marco Amatruda
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Aurelio Lo Buglio
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Michał Dobrakowski
- Department of Biochemistry, Medical University of Silesia, Zabrze 41-808, Poland
| | | | - Sławomir Kasperczyk
- Department of Biochemistry, Medical University of Silesia, Zabrze 41-808, Poland
| | - Gaetano Serviddio
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
| | - Gianluigi Vendemiale
- Department of Medical and Surgical Sciences, University of Foggia, Foggia 71122, Italy
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Brovold M, Keller D, Devarasetty M, Dominijanni A, Shirwaiker R, Soker S. Biofabricated 3D in vitro model of fibrosis-induced abnormal hepatoblast/biliary progenitors' expansion of the developing liver. Bioeng Transl Med 2021; 6:e10207. [PMID: 34589593 PMCID: PMC8459590 DOI: 10.1002/btm2.10207] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2020] [Revised: 11/25/2020] [Accepted: 11/29/2020] [Indexed: 11/19/2022] Open
Abstract
Congenital disorders of the biliary tract are the primary reason for pediatric liver failure and ultimately for pediatric liver transplant needs. Not all causes of these disorders are well understood, but it is known that liver fibrosis occurs in many of those afflicted. The goal of this study is to develop a simple yet robust model that recapitulates physico-mechanical and cellular aspects of fibrosis mediated via hepatic stellate cells (HSCs) and their effects on biliary progenitor cells. Liver organoids were fabricated by embedding various HSCs, with distinctive abilities to generate mild to severe fibrotic environments, together with undifferentiated liver progenitor cell line, HepaRG, within a collagen I hydrogel. The fibrotic state of each organoid was characterized by examination of extracellular matrix (ECM) remodeling through quantitative image analysis, rheometry, and qPCR. In tandem, the phenotype of the liver progenitor cell and cluster formation was assessed through histology. Activated HSCs (aHSCs) created a more severe fibrotic state, exemplified by a more highly contracted and rigid ECM, as well higher relative expression of TGF-β, TIMP-1, LOXL2, and COL1A2 as compared to immortalized HSCs (LX-2). Within the more severe fibrotic environment, generated by the aHSCs, higher Notch signaling was associated with an expansion of CK19+ cells as well as the formation of larger, more densely populated cell biliary like-clusters as compared to mild and non-fibrotic controls. The expansion of CK19+ cells, coupled with a severely fibrotic environment, are phenomena found within patients suffering from a variety of congenital liver disorders of the biliary tract. Thus, the model presented here can be utilized as a novel in vitro testing platform to test drugs and identify new targets that could benefit pediatric patients that suffer from the biliary dysgenesis associated with a multitude of congenital liver diseases.
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Affiliation(s)
- Matthew Brovold
- Wake Forest Institute for Regenerative MedicineWake Forest Baptist Medical Center, Medical Center BoulevardWinston‐SalemNorth CarolinaUSA
| | - Dale Keller
- Wake Forest Institute for Regenerative MedicineWake Forest Baptist Medical Center, Medical Center BoulevardWinston‐SalemNorth CarolinaUSA
| | - Mahesh Devarasetty
- Wake Forest Institute for Regenerative MedicineWake Forest Baptist Medical Center, Medical Center BoulevardWinston‐SalemNorth CarolinaUSA
| | - Anthony Dominijanni
- Wake Forest Institute for Regenerative MedicineWake Forest Baptist Medical Center, Medical Center BoulevardWinston‐SalemNorth CarolinaUSA
| | - Rohan Shirwaiker
- Department of Industrial and Systems EngineeringNorth Carolina State UniversityRaleighNorth CarolinaUSA
| | - Shay Soker
- Wake Forest Institute for Regenerative MedicineWake Forest Baptist Medical Center, Medical Center BoulevardWinston‐SalemNorth CarolinaUSA
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The Performance of HepG2 and HepaRG Systems through the Glass of Acetaminophen-Induced Toxicity. Life (Basel) 2021; 11:life11080856. [PMID: 34440600 PMCID: PMC8400973 DOI: 10.3390/life11080856] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2021] [Revised: 08/07/2021] [Accepted: 08/18/2021] [Indexed: 12/30/2022] Open
Abstract
Investigation of drug-induced liver injuries requires appropriate in vivo and in vitro toxicological model systems. In our study, an attempt was made to compare the hepatocarcinoma HepG2 and the stem cell-derived HepaRG cell lines both in two- and three-dimensional culture conditions to find the most suitable model. Comparison of the liver-specific characteristics of these models was performed via the extent and mechanism of acetaminophen (APAP)-induced hepatotoxicity. Investigating the detailed mechanism of APAP-induced hepatotoxicity, different specific cell death inhibitors were used: the pan-caspase inhibitor zVAD-fmk and dabrafenib significantly protected both cell lines from APAP-induced cell death. However, the known specific inhibitors of necroptosis (necrostatin-1 and MDIVI) were only effective in differentiated HepaRG, which suggest a differential execution of activated pathways in the two models. By applying 3D culture methods, CYP2E1 mRNA levels could be elevated, but we failed to achieve a significant increase in hepatocyte function; hence, the 3D cultivation especially in APAP toxicity studies is not necessarily worth the complicated maintenance. Based on our findings, the hepatocyte functions of HepaRG may stand between the properties of HepG2 cells and primary hepatocytes (PHHs). However, it should be noted that in contrast to PHHs having many limitations, HepaRG cells are relatively immortal, having a stable phenotype and CYP450 expression.
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Sonoi R, Hagihara Y. Tight junction stabilization prevents HepaRG cell death in drug-induced intrahepatic cholestasis. Biol Open 2021; 10:269189. [PMID: 34151938 PMCID: PMC8272035 DOI: 10.1242/bio.058606] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/25/2021] [Accepted: 05/18/2021] [Indexed: 01/11/2023] Open
Abstract
Entacapone (ENT), a catechol-O-methyltransferase inhibitor, causes liver injury by inducing bile canaliculi (BC) dilation through inhibition of the myosin light kinase pathway. Loss of tight junctions (TJs) induces hepatocyte depolarization, which causes bile secretory failure, leading to liver damage. To understand the influence of TJ structural changes as a consequence of BC dynamics, we compared the datasets of time-lapse and immunofluorescence images for TJ protein ZO-1 in hepatocytes cultured with ENT, forskolin (FOR), ENT/FOR, and those cultured without any drugs. Retrospective analysis revealed that the drastic change in BC behaviors caused TJ disruption and apoptosis in cells cultured with ENT. Exposure to FOR or sodium taurocholate facilitated TJ formation in the cells cultured with ENT and suppressed BC dynamic changes, leading to the inhibition of TJ disruption and apoptosis. Our findings clarify that hepatocyte TJ stabilization protects against cell death induced by BC disruption.
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Affiliation(s)
- Rie Sonoi
- Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan
| | - Yoshihisa Hagihara
- Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan
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35
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Ingelman-Sundberg M, Lauschke VM. 3D human liver spheroids for translational pharmacology and toxicology. Basic Clin Pharmacol Toxicol 2021; 130 Suppl 1:5-15. [PMID: 33872466 DOI: 10.1111/bcpt.13587] [Citation(s) in RCA: 28] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2021] [Revised: 04/12/2021] [Accepted: 04/13/2021] [Indexed: 12/14/2022]
Abstract
Drug development is a failure-prone endeavour, and more than 85% of drugs fail during clinical development, showcasing that current preclinical systems for compound selection are clearly inadequate. Liver toxicity remains a major reason for safety failures. Furthermore, all efforts to develop pharmacological therapies for a variety of chronic liver diseases, such as non-alcoholic steatohepatitis (NASH) and fibrosis, remain unsuccessful. Considering the time and expense of clinical trials, as well as the substantial burden on patients, new strategies are thus of paramount importance to increase clinical success rates. To this end, human liver spheroids are becoming increasingly utilized as they allow to preserve patient-specific phenotypes and functions for multiple weeks in culture. We here review the recent application of such systems for i) predictive and mechanistic analyses of drug hepatotoxicity, ii) the evaluation of hepatic disposition and metabolite formation of low clearance drugs and iii) the development of drugs for metabolic and infectious liver diseases, including NASH, fibrosis, malaria and viral hepatitis. We envision that with increasing dissemination, liver spheroids might become the new gold standard for such applications in translational pharmacology and toxicology.
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Affiliation(s)
| | - Volker M Lauschke
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
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36
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Gijbels E, Devisscher L, Vinken M. Testing in vitro tools for the prediction of cholestatic liver injury induced by non-pharmaceutical chemicals. Food Chem Toxicol 2021; 152:112165. [PMID: 33819548 DOI: 10.1016/j.fct.2021.112165] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/11/2021] [Revised: 03/15/2021] [Accepted: 03/29/2021] [Indexed: 02/07/2023]
Abstract
Bile acid accumulation and subsequent liver damage is a frequent adverse effect induced by drugs. Considerable efforts have therefore been focused on the introduction and characterization of tools that allow reliable prediction of this type of drug-induced liver injury. Among those are the cholestatic index and transcriptomic profiling, which are typically assessed in in vitro settings. The present study was set up to test the applicability of both tools to non-pharmaceutical compounds with cholestatic potential, including the industrial compound bis(2-ethylhexyl)phthalate, the cosmetic ingredients triclosan and octynoic acid, the herbicides paraquat and quizalofop-para-ethyl, and the food additives sunset yellow and tartrazine, in a human hepatoma cell culture model of cholestatic liver injury. The cholestatic index method showed cholestatic liability of sunset yellow, tartrazine and triclosan. Of those, tartrazine induced transcriptional changes reminiscent of the transcriptional profile of cholestatic drugs. Furthermore, a number of genes were found to be uniquely modulated by tartrazine, in accordance with the cholestatic drugs atazanavir, cyclosporin A and nefazodone, which may have potential as novel transcriptomic biomarkers of chemical-induced cholestatic liver injury. In conclusion, unambiguous identification of the non-pharmaceutical compounds tested in this study as inducers of cholestasis could not be achieved.
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Affiliation(s)
- Eva Gijbels
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium; Gut-Liver Immunopharmacology Unit, Basic and Applied Medical Sciences, Liver Research Center Ghent, Faculty of Medicine and Health Sciences, Ghent University, Corneel Heymanslaan 10, 9000, Ghent, Belgium
| | - Lindsey Devisscher
- Gut-Liver Immunopharmacology Unit, Basic and Applied Medical Sciences, Liver Research Center Ghent, Faculty of Medicine and Health Sciences, Ghent University, Corneel Heymanslaan 10, 9000, Ghent, Belgium
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
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37
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Zhuang X, Forde D, Tsukuda S, D'Arienzo V, Mailly L, Harris JM, Wing PAC, Borrmann H, Schilling M, Magri A, Rubio CO, Maidstone RJ, Iqbal M, Garzon M, Minisini R, Pirisi M, Butterworth S, Balfe P, Ray DW, Watashi K, Baumert TF, McKeating JA. Circadian control of hepatitis B virus replication. Nat Commun 2021; 12:1658. [PMID: 33712578 PMCID: PMC7955118 DOI: 10.1038/s41467-021-21821-0] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/07/2020] [Accepted: 02/02/2021] [Indexed: 02/06/2023] Open
Abstract
Chronic hepatitis B virus (HBV) infection is a major cause of liver disease and cancer worldwide for which there are no curative therapies. The major challenge in curing infection is eradicating or silencing the covalent closed circular DNA (cccDNA) form of the viral genome. The circadian factors BMAL1/CLOCK and REV-ERB are master regulators of the liver transcriptome and yet their role in HBV replication is unknown. We establish a circadian cycling liver cell-model and demonstrate that REV-ERB directly regulates NTCP-dependent hepatitis B and delta virus particle entry. Importantly, we show that pharmacological activation of REV-ERB inhibits HBV infection in vitro and in human liver chimeric mice. We uncover a role for BMAL1 to bind HBV genomes and increase viral promoter activity. Pharmacological inhibition of BMAL1 through REV-ERB ligands reduces pre-genomic RNA and de novo particle secretion. The presence of conserved E-box motifs among members of the Hepadnaviridae family highlight an evolutionarily conserved role for BMAL1 in regulating this family of small DNA viruses.
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Affiliation(s)
- Xiaodong Zhuang
- Nuffield Department of Medicine, University of Oxford, Oxford, UK.
| | - Donall Forde
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Senko Tsukuda
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- RIKEN Cluster for Pioneering Research, Wako, Japan
| | | | - Laurent Mailly
- University of Strasbourg and Inserm, UMR-S1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France
| | - James M Harris
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Peter A C Wing
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Helene Borrmann
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Mirjam Schilling
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - Andrea Magri
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | | | - Robert J Maidstone
- NIHR Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, UK
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, UK
| | - Mudassar Iqbal
- Division of Informatics, Imaging and Data Sciences, Faculty of Biology, Medicine and Health, University of Manchester, Manchester, UK
| | - Miguel Garzon
- Division of Pharmacy and Optometry, School of Health Sciences and Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK
| | - Rosalba Minisini
- Department of Translational Medicine, Università del Piemonte Orientale, Novara, Italy
| | - Mario Pirisi
- Department of Translational Medicine, Università del Piemonte Orientale, Novara, Italy
| | - Sam Butterworth
- Division of Pharmacy and Optometry, School of Health Sciences and Manchester Academic Health Sciences Centre, University of Manchester, Manchester, UK
| | - Peter Balfe
- Nuffield Department of Medicine, University of Oxford, Oxford, UK
| | - David W Ray
- NIHR Oxford Biomedical Research Centre, John Radcliffe Hospital, Oxford, UK
- Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, UK
| | - Koichi Watashi
- Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan
- RIKEN Cluster for Pioneering Research, Wako, Japan
- Department of Applied Biological Sciences, Tokyo University of Science Graduate School of Science and Technology, Japan and Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, Japan
| | - Thomas F Baumert
- University of Strasbourg and Inserm, UMR-S1110, Institut de Recherche sur les Maladies Virales et Hépatiques, Strasbourg, France
- Pôle Hépato-Digestif, Institut Hopitalo-Universitaire (IHU), Hopitaux Universitaire de Strasbourg, Strasbourg and Institut Universitaire de France, Paris, France
| | - Jane A McKeating
- Nuffield Department of Medicine, University of Oxford, Oxford, UK.
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Lauschke VM. Toxicogenomics of drug induced liver injury - from mechanistic understanding to early prediction. Drug Metab Rev 2021; 53:245-252. [PMID: 33683927 DOI: 10.1080/03602532.2021.1894571] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/09/2023]
Abstract
Despite rigorous preclinical testing, clinical attrition rates in drug development remain high with drug-induced liver injury (DILI) remaining one of the most frequent causes of project failures. To understand DILI mechanisms, major efforts are put into the development of physiologically relevant cell models and culture paradigms with the aim to enhance preclinical to clinical result translation. While the majority of toxicogenomic studies have been based on cell lines, there are emerging trends toward the predominant use of stem cell-derived organoids and primary human hepatocytes in complex 3D cell models. Such studies have been successful in disentangling diverse toxicity mechanisms, including genotoxicity, mitochondrial injury, steatogenesis and cholestasis and can aid in distinguishing hepatotoxic from nontoxic structural analogs. Furthermore, by leveraging inter-individual differences of cells from different donors, these approaches can emulate the complexity of polygenic risk scores, which facilitates personalized drug-specific DILI risk analyses. In summary, toxicogenomic studies into drug-induced hepatotoxicity have majorly contributed to our mechanistic understanding of DILI and the incorporation of organotypic human 3D liver models into the preclinical testing arsenal promises to enhance biological insights during drug discovery, increase confidence in preclinical safety and minimize the translational gap.
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Affiliation(s)
- Volker M Lauschke
- Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
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Pellerin M, Hirchaud E, Blanchard Y, Pavio N, Doceul V. Characterization of a Cell Culture System of Persistent Hepatitis E Virus Infection in the Human HepaRG Hepatic Cell Line. Viruses 2021; 13:406. [PMID: 33806591 PMCID: PMC8001476 DOI: 10.3390/v13030406] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2020] [Revised: 02/19/2021] [Accepted: 02/26/2021] [Indexed: 12/12/2022] Open
Abstract
Hepatitis E virus (HEV) is considered as an emerging global health problem. In most cases, hepatitis E is a self-limiting disease and the virus is cleared spontaneously without the need of antiviral therapy. However, immunocompromised individuals can develop chronic infection and liver fibrosis that can progress rapidly to cirrhosis and liver failure. The lack of efficient and relevant cell culture system and animal models has limited our understanding of the biology of HEV and the development of effective drugs for chronic cases. In the present study, we developed a model of persistent HEV infection in human hepatocytes in which HEV replicates efficiently. This HEV cell culture system is based on differentiated HepaRG cells infected with an isolate of HEV-3 derived from a patient suffering from acute hepatitis E. Efficient replication was maintained for several weeks to several months as well as after seven successive passages on HepaRG naïve cells. Moreover, after six passages onto HepaRG, we found that the virus was still infectious after oral inoculation into pigs. We also showed that ribavirin had an inhibitory effect on HEV replication in HepaRG. In conclusion, this system represents a relevant and efficient in vitro model of HEV replication that could be useful to study HEV biology and identify effective antiviral drugs against chronic HEV infection.
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Affiliation(s)
- Marie Pellerin
- UMR 1161 Virologie, INRAE, ANSES, Ecole Nationale Vétérinaire d’Alfort, Université Paris-Est, 94700 Maisons-Alfort, France; (M.P.); (N.P.)
| | - Edouard Hirchaud
- Agence Nationale de Sécurité Sanitaire, De L’environnement et du Travail (ANSES), Laboratory of Ploufragan-Plouzané-Niort, Viral Genetic and Biosafety (GVB) Unit, 22440 Ploufragan, France; (E.H.); (Y.B.)
| | - Yannick Blanchard
- Agence Nationale de Sécurité Sanitaire, De L’environnement et du Travail (ANSES), Laboratory of Ploufragan-Plouzané-Niort, Viral Genetic and Biosafety (GVB) Unit, 22440 Ploufragan, France; (E.H.); (Y.B.)
| | - Nicole Pavio
- UMR 1161 Virologie, INRAE, ANSES, Ecole Nationale Vétérinaire d’Alfort, Université Paris-Est, 94700 Maisons-Alfort, France; (M.P.); (N.P.)
| | - Virginie Doceul
- UMR 1161 Virologie, INRAE, ANSES, Ecole Nationale Vétérinaire d’Alfort, Université Paris-Est, 94700 Maisons-Alfort, France; (M.P.); (N.P.)
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40
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Sonoi R, Yamakawa T, Nakatani N, Kokubo M, Hagihara Y. Noninvasive Evaluation of HepaRG Aggregates during Drug‐Induced Intrahepatic Cholestasis Using Optical Coherence Tomography. Adv Biol (Weinh) 2021. [DOI: 10.1002/adbi.202000198] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022]
Affiliation(s)
- Rie Sonoi
- Biomedical Research Institute National Institute of Advanced Industrial Science and Technology 1‐8‐31 Midorigaoka Ikeda Osaka 563‐8577 Japan
| | - Takeshi Yamakawa
- R&D Department 1 Screen Holdings Co., Ltd. 322 Furukawa‐cho, Hazukashi Fushimi‐ku Kyoto 612‐8486 Japan
| | - Noriyuki Nakatani
- R&D Department 1 Screen Holdings Co., Ltd. 322 Furukawa‐cho, Hazukashi Fushimi‐ku Kyoto 612‐8486 Japan
| | - Masahiko Kokubo
- R&D Department 1 Screen Holdings Co., Ltd. 322 Furukawa‐cho, Hazukashi Fushimi‐ku Kyoto 612‐8486 Japan
| | - Yoshihisa Hagihara
- Biomedical Research Institute National Institute of Advanced Industrial Science and Technology 1‐8‐31 Midorigaoka Ikeda Osaka 563‐8577 Japan
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Yamazaki T, Tokiwa T. Elevated levels of expression of cytochrome P450 3A4 in a human liver epithelial cell line in differentiation-inducing conditions. Hum Cell 2021; 34:750-758. [PMID: 33495943 DOI: 10.1007/s13577-021-00487-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/18/2020] [Accepted: 01/08/2021] [Indexed: 10/22/2022]
Abstract
Cytochrome P450 (CYP) enzymes, especially CYP3A4 play a major role in the metabolism of xenobiotics in human liver. CYP3A4-expressing human liver or hepatoma cell lines may be good cell substitutes of human hepatocytes for drug metabolism studies. However, there are only a few cell lines expressing high levels of CYP3A4. The aim of this study is to investigate the expression of CYP3A4 and its mechanism in an immortalized non-tumorigenic human liver epithelial cell line, THLE-5b in differentiation-inducing conditions. When THLE-5b cells were cultivated in culture medium supplemented with hepatocytic differentiation-inducing factors, they showed hepatocytic morphology. In addition, elevated levels of expression not only of α1-antitrypsin (AAT) and albumin (ALB) mRNAs, but also of CYP3A4 mRNA, which are functional hepatocyte markers, were observed compared with the control. Among hepatocytic differentiation-inducing factors, dexamethasone (DEX) and insulin-transferrin-sodium selenite (ITS) seemed to be involved in elevation of expression of CYP3A4 mRNA. The mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) inhibitor U0126 or the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 reduced CYP3A4 mRNA levels of THLE-5b cells. Furthermore, the CpG site of the CYP3A4 promoter region in THLE-5b cells was found to be unmethylated, although in low CYP3A4-expressing HepG2 cells, the site was methylated. In conclusion, THLE-5b cells, which are unmethylated at the CpG site of the CYP3A4 promoter region, express CYP3A4 mRNA through the MEK/ERK1/2 and PI3K/Akt signaling pathways and acquire hepatocytic functions in differentiation-inducing conditions. Thus, THLE-5b cells could be a useful cell system for the study of drug metabolism.
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Affiliation(s)
- Taisuke Yamazaki
- Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, 3-4-4 Kita-shinagawa, Shinagawa-ku, Tokyo, 140-0001, Japan.
| | - Takayoshi Tokiwa
- Department of Liver Cell Biology, Kohno Clinical Medicine Research Institute, 3-4-4 Kita-shinagawa, Shinagawa-ku, Tokyo, 140-0001, Japan
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de Jong LM, Jiskoot W, Swen JJ, Manson ML. Distinct Effects of Inflammation on Cytochrome P450 Regulation and Drug Metabolism: Lessons from Experimental Models and a Potential Role for Pharmacogenetics. Genes (Basel) 2020; 11:genes11121509. [PMID: 33339226 PMCID: PMC7766585 DOI: 10.3390/genes11121509] [Citation(s) in RCA: 73] [Impact Index Per Article: 14.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2020] [Revised: 12/07/2020] [Accepted: 12/14/2020] [Indexed: 12/18/2022] Open
Abstract
Personalized medicine strives to optimize drug treatment for the individual patient by taking into account both genetic and non-genetic factors for drug response. Inflammation is one of the non-genetic factors that has been shown to greatly affect the metabolism of drugs—primarily through inhibition of cytochrome P450 (CYP450) drug-metabolizing enzymes—and hence contribute to the mismatch between the genotype predicted drug response and the actual phenotype, a phenomenon called phenoconversion. This review focuses on inflammation-induced drug metabolism alterations. In particular, we discuss the evidence assembled through human in-vitro models on the effect of inflammatory mediators on clinically relevant CYP450 isoform levels and their metabolizing capacity. We also present an overview of the current understanding of the mechanistic pathways via which inflammation in hepatocytes may modulate hepatic functions that are critical for drug metabolism. Furthermore, since large inter-individual variability in response to inflammation is observed in human in-vitro models and clinical studies, we evaluate the potential role of pharmacogenetic variability in the inflammatory signaling cascade and how this can modulate the outcome of inflammation on drug metabolism and response.
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Affiliation(s)
- Laura M. de Jong
- Division of BioTherapeutics, Leiden Academic Centre for Drug Research (LACDR), Leiden University, 2333 CC Leiden, The Netherlands; (L.M.d.J.); (W.J.)
| | - Wim Jiskoot
- Division of BioTherapeutics, Leiden Academic Centre for Drug Research (LACDR), Leiden University, 2333 CC Leiden, The Netherlands; (L.M.d.J.); (W.J.)
| | - Jesse J. Swen
- Department of Clinical Pharmacy & Toxicology, Leiden University Medical Center (LUMC), 2333 ZA Leiden, The Netherlands;
- Leiden Network for Personalised Therapeutics, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
| | - Martijn L. Manson
- Division of BioTherapeutics, Leiden Academic Centre for Drug Research (LACDR), Leiden University, 2333 CC Leiden, The Netherlands; (L.M.d.J.); (W.J.)
- Leiden Network for Personalised Therapeutics, Leiden University Medical Center, 2333 ZA Leiden, The Netherlands
- Correspondence:
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Effects of vanadium (sodium metavanadate) and aflatoxin-B1 on cytochrome p450 activities, DNA damage and DNA methylation in human liver cell lines. Toxicol In Vitro 2020; 70:105036. [PMID: 33164849 DOI: 10.1016/j.tiv.2020.105036] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Revised: 10/13/2020] [Accepted: 10/20/2020] [Indexed: 01/15/2023]
Abstract
Vanadium is considered as "possibly carcinogenic to humans" (V2O5, IARC Group 2B), yet uncertainties persist related to the toxicity mechanisms of the multiple forms of vanadium. Exposure to vanadium often co-occurs with other metals or with organic compounds that can be transformed by cytochrome p450 (CYP) enzymes into DNA-reactive carcinogens. Therefore, effects of a soluble form of vanadium (sodium metavanadate, NaVO3) and aflatoxin-B1 (AFB1) were tested separately and together, for induction of CYP activities, DNA damage (γH2AX and DNA alkaline unwinding assays), and DNA methylation changes (global genome and DNA repeats) in HepaRG or HepG2 liver cell lines. NaVO3 (≥ 2.3 μM) reduced CYP1A1 and CYP3A4 activities and induced DNA damage, butcaused important cell proliferation only in HepaRG cells. As a binary mixture, NaVO3 did not modify the effects of AFB1. There was no reproducible effect of NaVO3 (<21 μM) on DNA methylation in AluYb8, satellite-α, satellite-2, and by the luminometric methylation assay, but DNA methylation flow-cytometry signals in HepG2 cells (25-50 μM) increased at the G1 and G2 cell cycle phases. In conclusion, cell lines responded differently to NaVO3 supporting the importance of investigating more than one cell line, and a carcinogenic role of NaVO3 might reside at low concentrations by stimulating the proliferation of tumorigenic cells.
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Lucifora J, Michelet M, Salvetti A, Durantel D. Fast Differentiation of HepaRG Cells Allowing Hepatitis B and Delta Virus Infections. Cells 2020; 9:cells9102288. [PMID: 33066405 PMCID: PMC7602217 DOI: 10.3390/cells9102288] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2020] [Revised: 10/10/2020] [Accepted: 10/12/2020] [Indexed: 12/21/2022] Open
Abstract
HepaRG cells are liver bipotent progenitors acquiring hepatocytes features when differentiated in the presence of dimethylsulfoxide (DMSO). Differentiated HepaRG (dHepaRG) are considered the best surrogate model to primary human hepatocytes (PHH) and are susceptible to several hepatotropic viruses, including Hepatitis B Virus (HBV) and Hepatitis Delta Virus (HDV) infection. Despite these advantages, HepaRG cells are not widely used for the study of these two viruses because of their long differentiation process and their rather low and variable infection rates. Here, we tested the use of a cocktail of five chemicals (5C) combined or not with DMSO to accelerate the cells’ differentiation process. We found that NTCP-mediated HDV entry and replication are similar in HepaRG cells cultivated for only 1 week with 5C and DMSO or differentiated with the regular 4-week protocol. However, even though the NTCP-mediated HBV entry process seemed similar, cccDNA and subsequent HBV replication markers were lower in HepaRG cells cultivated for 1 week with 5C and DMSO compared to the regular differentiation protocol. In conclusion, we set up a new procedure allowing fast differentiation and efficient HDV-infection of HepaRG cells and identified differential culture conditions that may allow to decipher the mechanism behind the establishment of the HBV minichromosome.
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Kim J, Ryu B, Kim U, Kim CH, Hur GH, Kim CY, Park JH. Improved human hematopoietic reconstitution in HepaRG co-transplanted humanized NSG mice. BMB Rep 2020. [PMID: 32336318 PMCID: PMC7526976 DOI: 10.5483/bmbrep.2020.53.9.304] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Affiliation(s)
- Jin Kim
- Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 08826, Korea
| | - Bokyeong Ryu
- Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 08826, Korea
| | - Ukjin Kim
- Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 08826, Korea
| | - Chang-Hwan Kim
- The 4th R&D Institute-6, Agency for Defense Development, Daejeon 34186, Korea
| | - Gyeung-Haeng Hur
- The 4th R&D Institute-6, Agency for Defense Development, Daejeon 34186, Korea
| | - C-Yoon Kim
- Stem Cell Biology, School of Medicine, Konkuk University, Seoul 05030, Korea
| | - Jae-Hak Park
- Department of Laboratory Animal Medicine, College of Veterinary Medicine, Seoul National University, Seoul 08826, Korea
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46
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Dolicka D, Sobolewski C, Gjorgjieva M, Correia de Sousa M, Berthou F, De Vito C, Colin DJ, Bejuy O, Fournier M, Maeder C, Blackshear PJ, Rubbia-Brandt L, Foti M. Tristetraprolin Promotes Hepatic Inflammation and Tumor Initiation but Restrains Cancer Progression to Malignancy. Cell Mol Gastroenterol Hepatol 2020; 11:597-621. [PMID: 32987153 PMCID: PMC7806869 DOI: 10.1016/j.jcmgh.2020.09.012] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/18/2020] [Revised: 09/21/2020] [Accepted: 09/22/2020] [Indexed: 02/07/2023]
Abstract
BACKGROUND & AIMS Tristetraprolin (TTP) is a key post-transcriptional regulator of inflammatory and oncogenic transcripts. Accordingly, TTP was reported to act as a tumor suppressor in specific cancers. Herein, we investigated how TTP contributes to the development of liver inflammation and fibrosis, which are key drivers of hepatocarcinogenesis, as well as to the onset and progression of hepatocellular carcinoma (HCC). METHODS TTP expression was investigated in mouse/human models of hepatic metabolic diseases and cancer. The role of TTP in nonalcoholic steatohepatitis and HCC development was further examined through in vivo/vitro approaches using liver-specific TTP knockout mice and a panel of hepatic cancer cells. RESULTS Our data demonstrate that TTP loss in vivo strongly restrains development of hepatic steatosis and inflammation/fibrosis in mice fed a methionine/choline-deficient diet, as well as HCC development induced by the carcinogen diethylnitrosamine. In contrast, low TTP expression fostered migration and invasion capacities of in vitro transformed hepatic cancer cells likely by unleashing expression of key oncogenes previously associated with these cancerous features. Consistent with these data, TTP was significantly down-regulated in high-grade human HCC, a feature further correlating with poor clinical prognosis. Finally, we uncover hepatocyte nuclear factor 4 alpha and early growth response 1, two key transcription factors lost with hepatocyte dedifferentiation, as key regulators of TTP expression. CONCLUSIONS Although TTP importantly contributes to hepatic inflammation and cancer initiation, its loss with hepatocyte dedifferentiation fosters cancer cells migration and invasion. Loss of TTP may represent a clinically relevant biomarker of high-grade HCC associated with poor prognosis.
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MESH Headings
- Animals
- Carcinogenesis/genetics
- Carcinogenesis/immunology
- Carcinogenesis/pathology
- Carcinoma, Hepatocellular/genetics
- Carcinoma, Hepatocellular/immunology
- Carcinoma, Hepatocellular/mortality
- Carcinoma, Hepatocellular/pathology
- Cell Line, Tumor
- Datasets as Topic
- Diethylnitrosamine/administration & dosage
- Diethylnitrosamine/toxicity
- Down-Regulation
- Female
- Gene Expression Regulation, Neoplastic/immunology
- Hepatocytes
- Humans
- Liver/immunology
- Liver/pathology
- Liver Cirrhosis/genetics
- Liver Cirrhosis/immunology
- Liver Cirrhosis/pathology
- Liver Neoplasms/genetics
- Liver Neoplasms/immunology
- Liver Neoplasms/mortality
- Liver Neoplasms/pathology
- Liver Neoplasms, Experimental/chemistry
- Liver Neoplasms, Experimental/genetics
- Liver Neoplasms, Experimental/immunology
- Liver Neoplasms, Experimental/pathology
- Male
- Mice
- Non-alcoholic Fatty Liver Disease
- Primary Cell Culture
- Prognosis
- RNA-Seq
- Survival Analysis
- Tristetraprolin/genetics
- Tristetraprolin/metabolism
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Affiliation(s)
- Dobrochna Dolicka
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland
| | - Cyril Sobolewski
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland
| | - Monika Gjorgjieva
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland
| | - Marta Correia de Sousa
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland
| | - Flavien Berthou
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland
| | - Claudio De Vito
- Division of Clinical Pathology, University Hospitals, Geneva, Switzerland
| | - Didier J Colin
- Centre for Biomedical Imaging and Preclinical Imaging Platform, University of Geneva, Geneva, Switzerland
| | - Olivia Bejuy
- Centre for Biomedical Imaging and Preclinical Imaging Platform, University of Geneva, Geneva, Switzerland
| | - Margot Fournier
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland
| | - Christine Maeder
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland
| | - Perry J Blackshear
- Laboratory of Signal Transduction, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina
| | | | - Michelangelo Foti
- Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, Geneva, Switzerland; Translational Research Centre in Onco-haematology, Faculty of Medicine, University of Geneva, Switzerland.
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Burenina OY, Zatsepin TS, Kim EF, Metelin AV, Skvortsov DA, Rubtsova MP, Dontsova OA. Comparative Analysis of Long Noncoding RNA Expression in Human Hepatocyte Cell Lines and Liver. DOKL BIOCHEM BIOPHYS 2020; 493:181-184. [PMID: 32894460 DOI: 10.1134/s1607672920040043] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2020] [Revised: 03/23/2020] [Accepted: 03/23/2020] [Indexed: 12/29/2022]
Abstract
Long noncoding RNAs (lncRNAs) are promising biomarkers and potential targets for liver cancer therapy. Stable hepatocyte lines are used in vitro to investigate functions of lncRNAs which amount in cell fluctuates during carcinogenesis. For the first time we compared gene expression of known lncRNAs in human conditional normal liver cells HepaRG and cancer cell lines Huh7 and HepG2. We showed that relative amounts of these lncRNAs in HepaRG are close to analogous variables measured for liver samples from healthy donors. Obtained data demonstrate exclusive peculiarities of HepaRG and confirm its reasonable application as a model of normal human hepatocytes for studying functions of lncRNAs.
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Affiliation(s)
- O Y Burenina
- Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russia.
| | - T S Zatsepin
- Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russia.,Belozersky Institute of Physico-Chemical Biology and Chemistry Department, Moscow State University, Moscow, Russia
| | - E F Kim
- Petrovsky National Research Centre of Surgery, Moscow, Russia
| | - A V Metelin
- Petrovsky National Research Centre of Surgery, Moscow, Russia
| | - D A Skvortsov
- Belozersky Institute of Physico-Chemical Biology and Chemistry Department, Moscow State University, Moscow, Russia.,Faculty of Biology and Biotechnologies, Higher School of Economics, Moscow, Russia
| | - M P Rubtsova
- Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russia.,Belozersky Institute of Physico-Chemical Biology and Chemistry Department, Moscow State University, Moscow, Russia
| | - O A Dontsova
- Center of Life Sciences, Skolkovo Institute of Science and Technology, Moscow, Russia.,Belozersky Institute of Physico-Chemical Biology and Chemistry Department, Moscow State University, Moscow, Russia
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48
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Cooreman A, Van Campenhout R, Crespo Yanguas S, Gijbels E, Leroy K, Pieters A, Tabernilla A, Van Brantegem P, Annaert P, Cogliati B, Vinken M. Cholestasis Differentially Affects Liver Connexins. Int J Mol Sci 2020; 21:E6534. [PMID: 32906817 PMCID: PMC7116118 DOI: 10.3390/ijms21186534] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/03/2020] [Revised: 08/26/2020] [Accepted: 09/05/2020] [Indexed: 12/11/2022] Open
Abstract
Connexins are goal keepers of tissue homeostasis, including in the liver. As a result, they are frequently involved in disease. The current study was set up to investigate the effects of cholestatic disease on the production of connexin26, connexin32 and connexin43 in the liver. For this purpose, bile duct ligation, a well-known trigger of cholestatic liver injury, was applied to mice. In parallel, human hepatoma HepaRG cell cultures were exposed to cholestatic drugs and bile acids. Samples from both the in vivo and in vitro settings were subsequently subjected to assessment of mRNA and protein quantities as well as to in situ immunostaining. While the outcome of cholestasis on connexin26 and connexin43 varied among experimental settings, a more generalized repressing effect was seen for connexin32. This has also been observed in many other liver pathologies and could suggest a role for connexin32 as a robust biomarker of liver disease and toxicity.
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Affiliation(s)
- Axelle Cooreman
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (A.C.); (R.V.C.); (S.C.Y.); (E.G.); (K.L.); (A.P.); (A.T.)
| | - Raf Van Campenhout
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (A.C.); (R.V.C.); (S.C.Y.); (E.G.); (K.L.); (A.P.); (A.T.)
| | - Sara Crespo Yanguas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (A.C.); (R.V.C.); (S.C.Y.); (E.G.); (K.L.); (A.P.); (A.T.)
| | - Eva Gijbels
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (A.C.); (R.V.C.); (S.C.Y.); (E.G.); (K.L.); (A.P.); (A.T.)
| | - Kaat Leroy
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (A.C.); (R.V.C.); (S.C.Y.); (E.G.); (K.L.); (A.P.); (A.T.)
| | - Alanah Pieters
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (A.C.); (R.V.C.); (S.C.Y.); (E.G.); (K.L.); (A.P.); (A.T.)
| | - Andrés Tabernilla
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (A.C.); (R.V.C.); (S.C.Y.); (E.G.); (K.L.); (A.P.); (A.T.)
| | - Pieter Van Brantegem
- Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, 3000 Leuven, Belgium; (P.V.B.); (P.A.)
| | - Pieter Annaert
- Drug Delivery and Disposition, KU Leuven Department of Pharmaceutical and Pharmacological Sciences, 3000 Leuven, Belgium; (P.V.B.); (P.A.)
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo, São Paulo 05508-270, Brazil;
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (A.C.); (R.V.C.); (S.C.Y.); (E.G.); (K.L.); (A.P.); (A.T.)
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49
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Sonoi R, Hagihara Y. Switching of cell fate through the regulation of cell growth during drug-induced intrahepatic cholestasis. J Biosci Bioeng 2020; 130:659-665. [PMID: 32868186 DOI: 10.1016/j.jbiosc.2020.08.004] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/09/2020] [Revised: 07/28/2020] [Accepted: 08/02/2020] [Indexed: 12/12/2022]
Abstract
Understanding the fundamental mechanisms that govern the fate of cells during drug-induced intrahepatic cholestasis provides strategies for the establishment of evaluation methods for drug screening. In the present study, the aggregates of a differentiated human hepatic cell line, HepaRG, were incubated in medium with Y27632 or bosentan to clarify the changes in the behavior of bile canaliculi (BC) with the growth of cells during drug-induced intrahepatic cholestasis. With elapsed exposure time, the aggregates in the culture with bosentan caused the dilation of BC, and the hepatocytes ultimately exhibited apoptotic death after the disruption of BC. Y27632 caused the disruption of BC in the aggregates after dilation. However, there was no change in the number of cells within the aggregates in the culture with Y27632, in spite of its cytotoxicity. After 144 h from the start of Y27632 exposure, the aggregates showed the rearrangement of BC. To inhibit cell division, the aggregates exposed to Y27632, which exhibited disruption of BC, were treated with mitomycin C for 2 h and continuously exposed to Y27632. The inhibition of cell division could not induce the rearrangement of BC within these aggregates, which was similar to the phenomenon observed in the aggregates exposed to bosentan. These findings indicate that growth is an important factor that influences the switching of cell fate toward survival or death in drug-induced intrahepatic cholestasis process. Thus, the autoregulation of growth is a major contributor to the rearrangement of BC within aggregates.
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Affiliation(s)
- Rie Sonoi
- Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan.
| | - Yoshihisa Hagihara
- Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan
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50
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Svobodová J, Procházková J, Kabátková M, Krkoška M, Šmerdová L, Líbalová H, Topinka J, Kléma J, Kozubík A, Machala M, Vondráček J. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Disrupts Control of Cell Proliferation and Apoptosis in a Human Model of Adult Liver Progenitors. Toxicol Sci 2020; 172:368-384. [PMID: 31536130 DOI: 10.1093/toxsci/kfz202] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023] Open
Abstract
The aryl hydrocarbon receptor (AhR) activation has been shown to alter proliferation, apoptosis, or differentiation of adult rat liver progenitors. Here, we investigated the impact of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated AhR activation on a human model of bipotent liver progenitors, undifferentiated HepaRG cells. We used both intact undifferentiated HepaRG cells, and the cells with silenced Hippo pathway effectors, yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), which play key role(s) in tissue-specific progenitor cell self-renewal and expansion, such as in liver, cardiac, or respiratory progenitors. TCDD induced cell proliferation in confluent undifferentiated HepaRG cells; however, following YAP, and, in particular, double YAP/TAZ knockdown, TCDD promoted induction of apoptosis. These results suggested that, unlike in mature hepatocytes, or hepatocyte-like cells, activation of the AhR may sensitize undifferentiated HepaRG cells to apoptotic stimuli. Induction of apoptosis in cells with silenced YAP/TAZ was associated with upregulation of death ligand TRAIL, and seemed to involve both extrinsic and mitochondrial apoptosis pathways. Global gene expression analysis further suggested that TCDD significantly altered expression of constituents and/or transcriptional targets of signaling pathways participating in control of expansion or differentiation of liver progenitors, including EGFR, Wnt/β-catenin, or tumor growth factor-β signaling pathways. TCDD significantly upregulated cytosolic proapoptotic protein BMF (Bcl-2 modifying factor) in HepaRG cells, which could be linked with an enhanced sensitivity of TCDD-treated cells to apoptosis. Our results suggest that, in addition to promotion of cell proliferation and alteration of signaling pathways controlling expansion of human adult liver progenitors, AhR ligands may also sensitize human liver progenitor cells to apoptosis.
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Affiliation(s)
- Jana Svobodová
- Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno 61265, Czech Republic
- Department of Experimental Biology, Faculty of Science, Masaryk University, Brno 61137, Czech Republic
| | - Jiřina Procházková
- Department of Chemistry and Toxicology, Veterinary Research Institute, Brno 62100, Czech Republic
| | - Markéta Kabátková
- Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno 61265, Czech Republic
| | - Martin Krkoška
- Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno 61265, Czech Republic
- Department of Experimental Biology, Faculty of Science, Masaryk University, Brno 61137, Czech Republic
| | - Lenka Šmerdová
- Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno 61265, Czech Republic
| | - Helena Líbalová
- Department of Genetic Ecotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague 14220, Czech Republic
| | - Jan Topinka
- Department of Genetic Ecotoxicology, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague 14220, Czech Republic
| | - Jiří Kléma
- Department of Computer Science, Czech Technical University, Prague 12135, Czech Republic
| | - Alois Kozubík
- Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno 61265, Czech Republic
| | - Miroslav Machala
- Department of Chemistry and Toxicology, Veterinary Research Institute, Brno 62100, Czech Republic
| | - Jan Vondráček
- Department of Cytokinetics, Institute of Biophysics of the Czech Academy of Sciences, Brno 61265, Czech Republic
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