|
1
|
Kwon Y, Gottmann P, Wang S, Tissink J, Motzler K, Sekar R, Albrecht W, Cadenas C, Hengstler JG, Schürmann A, Zeigerer A. Induction of steatosis in primary human hepatocytes recapitulates key pathophysiological aspects of metabolic dysfunction-associated steatotic liver disease. J Hepatol 2025; 82:18-27. [PMID: 38977136 DOI: 10.1016/j.jhep.2024.06.040] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Revised: 06/15/2024] [Accepted: 06/29/2024] [Indexed: 07/10/2024]
Abstract
BACKGROUND & AIMS Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common cause of chronic liver disease. Owing to limited available treatment options, novel pre-clinical models for target selection and drug validation are warranted. We have established and extensively characterized a primary human steatotic hepatocyte in vitro model system that could guide the development of treatment strategies for MASLD. METHODS Cryopreserved primary human hepatocytes from five donors varying in sex and ethnicity were cultured with free fatty acids in a 3D collagen sandwich for 7 days and the development of MASLD was followed by assessing classical hepatocellular functions. As proof of concept, the effects of the drug firsocostat (GS-0976) on in vitro MASLD phenotypes were evaluated. RESULTS Incubation with free fatty acids induced steatosis, insulin resistance, mitochondrial dysfunction, inflammation, and alterations in prominent human gene signatures similar to patients with MASLD, indicating the recapitulation of human MASLD in this system. The application of firsocostat rescued clinically observed fatty liver disease pathologies, highlighting the ability of the in vitro system to test the efficacy and potentially characterize the mode of action of drug candidates. CONCLUSIONS Altogether, our human MASLD in vitro model system could guide the development and validation of novel targets and drugs for the treatment of MASLD. IMPACT AND IMPLICATIONS Due to low drug efficacy and high toxicity, clinical treatment options for metabolic dysfunction-associated steatotic liver disease (MASLD) are currently limited. To facilitate earlier stop-go decisions in drug development, we have established a primary human steatotic hepatocyte in vitro model. As the model recapitulates clinically relevant MASLD characteristics at high phenotypic resolution, it can serve as a pre-screening platform and guide target identification and validation in MASLD therapy.
Collapse
Affiliation(s)
- Yun Kwon
- Institute for Diabetes and Cancer, Helmholtz Center Munich, 85764 Neuherberg, Germany, Joint Heidelberg-IDC Translational Diabetes Program, Inner Medicine 1, Heidelberg University Hospital, Heidelberg, Germany; German Center for Diabetes Research (DZD), Neuherberg, Germany; European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany
| | - Pascal Gottmann
- German Center for Diabetes Research (DZD), Neuherberg, Germany; German Institute of Human Nutrition (DIfE), Department of Experimental Diabetology, Nuthetal, Germany
| | - Surui Wang
- Institute for Diabetes and Cancer, Helmholtz Center Munich, 85764 Neuherberg, Germany, Joint Heidelberg-IDC Translational Diabetes Program, Inner Medicine 1, Heidelberg University Hospital, Heidelberg, Germany; German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Joel Tissink
- Institute for Diabetes and Cancer, Helmholtz Center Munich, 85764 Neuherberg, Germany, Joint Heidelberg-IDC Translational Diabetes Program, Inner Medicine 1, Heidelberg University Hospital, Heidelberg, Germany; German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Karsten Motzler
- Institute for Diabetes and Cancer, Helmholtz Center Munich, 85764 Neuherberg, Germany, Joint Heidelberg-IDC Translational Diabetes Program, Inner Medicine 1, Heidelberg University Hospital, Heidelberg, Germany; German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Revathi Sekar
- Institute for Diabetes and Cancer, Helmholtz Center Munich, 85764 Neuherberg, Germany, Joint Heidelberg-IDC Translational Diabetes Program, Inner Medicine 1, Heidelberg University Hospital, Heidelberg, Germany; German Center for Diabetes Research (DZD), Neuherberg, Germany
| | - Wiebke Albrecht
- Leibniz Research Centre for Working Environment and Human Factors (IfADo), Department of Toxicology, Dortmund, Germany
| | - Cristina Cadenas
- Leibniz Research Centre for Working Environment and Human Factors (IfADo), Department of Toxicology, Dortmund, Germany
| | - Jan G Hengstler
- Leibniz Research Centre for Working Environment and Human Factors (IfADo), Department of Toxicology, Dortmund, Germany
| | - Annette Schürmann
- German Center for Diabetes Research (DZD), Neuherberg, Germany; German Institute of Human Nutrition (DIfE), Department of Experimental Diabetology, Nuthetal, Germany
| | - Anja Zeigerer
- Institute for Diabetes and Cancer, Helmholtz Center Munich, 85764 Neuherberg, Germany, Joint Heidelberg-IDC Translational Diabetes Program, Inner Medicine 1, Heidelberg University Hospital, Heidelberg, Germany; German Center for Diabetes Research (DZD), Neuherberg, Germany; European Center for Angioscience (ECAS), Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany.
| |
Collapse
|
|
2
|
Shitova M, Alpeeva E, Vorotelyak E. Review of hTERT-Immortalized Cells: How to Assess Immortality and Confirm Identity. Int J Mol Sci 2024; 25:13054. [PMID: 39684765 DOI: 10.3390/ijms252313054] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2024] [Revised: 11/22/2024] [Accepted: 11/29/2024] [Indexed: 12/18/2024] Open
Abstract
Cell immortalization has an important role in scientific research, as well as increasing significance in the context of cell therapy and biotechnology. Over the years, many immortalized cell lines have been produced using human telomerase reverse transcriptase (hTERT) alone or in a combination with viral oncogenes. Different hTERT-immortalized cells are commercially available, and numerous papers about obtaining immortalized cell lines have also been published. However, no specific list of characteristics that need to be checked to confirm successful immortalization exists. Most researchers evaluate only a few parameters, while different articles contain various opinions on the assessment of these characteristics. Results also vary significantly between different cell types, which have their own traits depending on their origin and functions. In the current paper, we raise these questions and discuss controversial issues concerning currently available testing methods for immortalization evaluation and the value and the limitations of the approaches. In addition, we propose a protocol for evaluation of hTERT immortalization success consisting of the following important steps: the assessment of the proliferation rate and dividing capacity, cell morphology, phenotype, karyotype stability, telomerase activity, the expression of cell-specific markers, and tumorigenicity. To our opinion, the hTERT expression level, telomere length, and senescence-associated β-galactosidase staining are controversial with regard to the implemented methods, so these parameters may be optional. For all the evaluation steps, we recommend to pay attention to the necessity of comparing the traits of the obtained immortalized and parent cells.
Collapse
Affiliation(s)
- Maria Shitova
- Laboratory of Cell Biology, N.K. Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Vavilov Street 26, 119334 Moscow, Russia
| | - Elena Alpeeva
- Laboratory of Cell Biology, N.K. Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Vavilov Street 26, 119334 Moscow, Russia
| | - Ekaterina Vorotelyak
- Laboratory of Cell Biology, N.K. Koltzov Institute of Developmental Biology of Russian Academy of Sciences, Vavilov Street 26, 119334 Moscow, Russia
| |
Collapse
|
|
3
|
Wani SI, Mir TA, Nakamura M, Tsuchiya T, Alzhrani A, Iwanaga S, Arai K, Alshehri EA, Shamma T, Obeid DA, Chinnappan R, Assiri AM, Yaqinuddin A, Vashist YK, Broering DC. A review of current state-of-the-art materiobiology and technological approaches for liver tissue engineering. BIOPRINTING 2024; 42:e00355. [DOI: 10.1016/j.bprint.2024.e00355] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/20/2025]
|
|
4
|
Smiriglia A, Lorito N, Serra M, Perra A, Morandi A, Kowalik MA. Sex difference in liver diseases: How preclinical models help to dissect the sex-related mechanisms sustaining NAFLD and hepatocellular carcinoma. iScience 2023; 26:108363. [PMID: 38034347 PMCID: PMC10682354 DOI: 10.1016/j.isci.2023.108363] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/02/2023] Open
Abstract
Only a few preclinical findings are confirmed in the clinic, posing a critical issue for clinical development. Therefore, identifying the best preclinical models can help to dissect molecular and mechanistic insights into liver disease pathogenesis while being clinically relevant. In this context, the sex relevance of most preclinical models has been only partially considered. This is particularly significant in NAFLD and HCC, which have a higher prevalence in men when compared to pre-menopause women but not to those in post-menopausal status, suggesting a role for sex hormones in the pathogenesis of the diseases. This review gathers the sex-relevant findings and the available preclinical models focusing on both in vitro and in vivo studies and discusses the potential implications and perspectives of introducing the sex effect in the selection of the best preclinical model. This is a critical aspect that would help to tailor personalized therapies based on sex.
Collapse
Affiliation(s)
- Alfredo Smiriglia
- Department of Experimental and Clinical Biomedical Sciences, University of Florence, 50134 Florence, Italy
| | - Nicla Lorito
- Department of Experimental and Clinical Biomedical Sciences, University of Florence, 50134 Florence, Italy
| | - Marina Serra
- Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato, Italy
| | - Andrea Perra
- Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato, Italy
| | - Andrea Morandi
- Department of Experimental and Clinical Biomedical Sciences, University of Florence, 50134 Florence, Italy
| | - Marta Anna Kowalik
- Department of Biomedical Sciences, University of Cagliari, 09042 Monserrato, Italy
| |
Collapse
|
|
5
|
Krylov D, Rodimova S, Karabut M, Kuznetsova D. Experimental Models for Studying Structural and Functional State of the Pathological Liver (Review). Sovrem Tekhnologii Med 2023; 15:65-82. [PMID: 38434194 PMCID: PMC10902899 DOI: 10.17691/stm2023.15.4.06] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2023] [Indexed: 03/05/2024] Open
Abstract
Liver pathologies remain one of the leading causes of mortality worldwide. Despite a high prevalence of liver diseases, the possibilities of diagnosing, prognosing, and treating non-alcoholic and alcoholic liver diseases still have a number of limitations and require the development of new methods and approaches. In laboratory studies, various models are used to reconstitute the pathological conditions of the liver, including cell cultures, spheroids, organoids, microfluidic systems, tissue slices. We reviewed the most commonly used in vivo, in vitro, and ex vivo models for studying non-alcoholic fatty liver disease and alcoholic liver disease, toxic liver injury, and fibrosis, described their advantages, limitations, and prospects for use. Great emphasis was placed on the mechanisms of development of pathological conditions in each model, as well as the assessment of the possibility of reconstructing various key aspects of pathogenesis for all these pathologies. There is currently no consensus on the choice of the most adequate model for studying liver pathology. The choice of a certain effective research model is determined by the specific purpose and objectives of the experiment.
Collapse
Affiliation(s)
- D.P. Krylov
- Laboratory Assistant, Scientific Laboratory of Molecular Biotechnologies, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia; Student, Institute of Biology and Biomedicine; National Research Lobachevsky State University of Nizhny Novgorod, 23 Prospekt Gagarina, Nizhny Novgorod, 603022, Russia
| | - S.A. Rodimova
- Junior Researcher, Laboratory of Regenerative Medicine, Scientific Laboratory of Molecular Biotechnologies, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia
| | - M.M. Karabut
- Researcher, Laboratory of Genomics of Adaptive Antitumor Immunity, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia
| | - D.S. Kuznetsova
- Head of Laboratory of Molecular Biotechnologies, Research Institute of Experimental Oncology and Biomedical Technologies; Privolzhsky Research Medical University, 10/1 Minin and Pozharsky Square, Nizhny Novgorod, 603005, Russia; Head of the Research Laboratory for Molecular Genetic Researches, Institute of Clinical Medicine; National Research Lobachevsky State University of Nizhny Novgorod, 23 Prospekt Gagarina, Nizhny Novgorod, 603022, Russia
| |
Collapse
|
|
6
|
Chiu CH. CRISPR/Cas9 genetic screens in hepatocellular carcinoma gene discovery. CURRENT RESEARCH IN BIOTECHNOLOGY 2023; 5:100127. [DOI: 10.1016/j.crbiot.2023.100127] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2025] Open
|
|
7
|
Shivaram S, Gao H, Qin S, Liu D, Weinshilboum RM, Wang L. Cytochrome P450 Transcriptional Regulation by Testis-Specific Y-Encoded-Like Protein: Identification of Novel Upstream Transcription Factors. Drug Metab Dispos 2023; 51:1-7. [PMID: 36153008 PMCID: PMC9832376 DOI: 10.1124/dmd.122.000945] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2022] [Revised: 07/05/2022] [Accepted: 07/05/2022] [Indexed: 01/14/2023] Open
Abstract
Cytochrome P450s (CYPs) display significant inter-individual variation in expression, much of which remains unexplained by known CYP single-nucleotide polymorphisms (SNPs). Testis-specific Y-encoded-like proteins (TSPYLs) are transcriptional regulators for several drug-metabolizing CYPs including CYP3A4 However, transcription factors (TFs) that might influence CYP expression through an effect on TSPYL expression are unknown. Therefore, we studied regulators of TSPYL expression in hepatic cell lines and their possible SNP-dependent variation. Specifically, we identified candidate TFs that might influence TSPYL expression using the ENCODE ChIPseq database. Subsequently, the expression of TSPYL1/2/4 as well as that of selected CYP targets for TSPYL regulation were assayed in hepatic cell lines before and after knockdown of TFs that might influence CYP expression through TSPYL-dependent mechanisms. Those results were confirmed by studies of TF binding to TSPYL1/2/4 gene promoter regions. In hepatic cell lines, knockdown of the REST and ZBTB7A TFs resulted in decreased TSPYL1 and TSPYL4 expression and increased CYP3A4 expression, changes reversed by TSPYL1/4 overexpression. Potential binding sites for REST and ZBTB7A on the promoters of TSPYL1 and TSPYL4 were confirmed by chromatin immunoprecipitation. Finally, common SNP variants in upstream binding sites on the TSPYL1/4 promoters were identified and luciferase reporter constructs confirmed SNP-dependent modulation of TSPYL1/4 gene transcription. In summary, we identified REST and ZBTB7A as regulators of the expression of TSPYL genes which themselves can contribute to regulation of CYP expression and-potentially-of drug metabolism. SNP-dependent modulation of TSPYL transcription may contribute to individual variation in both CYP expression and-downstream-drug response phenotypes. SIGNIFICANCE STATEMENT: Testis-specific Y-encoded-like proteins (TSPYLs) are transcriptional regulators of cytochrome P450 (CYP) gene expression. Here, we report that variation in TSPYL expression as a result of the effects of genetically regulated TSPYL transcription factors is an additional factor that could result in downstream variation in CYP expression and potentially, as a result, variation in drug biotransformation.
Collapse
Affiliation(s)
- Suganti Shivaram
- Division of Clinical Pharmacology, Department of Molecular Pharmacology and Therapeutics, Mayo Clinic, Rochester, Minnesota
| | - Huanyao Gao
- Division of Clinical Pharmacology, Department of Molecular Pharmacology and Therapeutics, Mayo Clinic, Rochester, Minnesota
| | - Sisi Qin
- Division of Clinical Pharmacology, Department of Molecular Pharmacology and Therapeutics, Mayo Clinic, Rochester, Minnesota
| | - Duan Liu
- Division of Clinical Pharmacology, Department of Molecular Pharmacology and Therapeutics, Mayo Clinic, Rochester, Minnesota
| | - Richard M Weinshilboum
- Division of Clinical Pharmacology, Department of Molecular Pharmacology and Therapeutics, Mayo Clinic, Rochester, Minnesota
| | - Liewei Wang
- Division of Clinical Pharmacology, Department of Molecular Pharmacology and Therapeutics, Mayo Clinic, Rochester, Minnesota
| |
Collapse
|
|
8
|
Shedding light on non-alcoholic fatty liver disease: Pathogenesis, molecular mechanisms, models, and emerging therapeutics. Life Sci 2022; 312:121185. [PMID: 36375569 DOI: 10.1016/j.lfs.2022.121185] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2022] [Revised: 11/07/2022] [Accepted: 11/08/2022] [Indexed: 11/13/2022]
Abstract
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder globally impacting an estimated 25% of the population associated with severe consequences such as cirrhosis, hepatocellular carcinoma (HCC), and overall mortality. Fatty liver disease is triggered through multiple pathways, but the most prominent cause is either diabetes or obesity, or a combination of both. Therefore, hepatic glucose, insulin and fatty acid signaling becomes a dire need to understand which is well elaborated in this review. This review summarizes the popular two-hit pathogenesis of NAFLD, the molecular mechanisms underlying hepatic insulin resistance. As fatty liver disease gets advanced, it requires in-vitro as well as in-vivo models closer to disease progression in humans for better understanding the pathological state and identifying a novel therapeutic target. This review summarizes in-vitro (2D cell-culture/co-culture, 3D spheroid/organoid/liver-on-a-chip) models as well as in-vivo (genetically/dietary/chemically induced fatty liver disease) research models. Fatty liver disease research has gathered lots of attention recently since there is no FDA approved therapy available so far. However, there have been numerous promising targets to treat fatty liver disease including potential therapeutic targets under clinical trials are listed in this review.
Collapse
|
|
9
|
Miranda-Velez M, Sarker GS, Ramisetty P, Geden S, Bartolomeu Halicki PC, Annamalai T, Tse-Dinh YC, Rohde KH, Moon JH. Proton Motive Force-Disrupting Antimycobacterial Guanylurea Polymer. Biomacromolecules 2022; 23:4668-4677. [DOI: 10.1021/acs.biomac.2c00902] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/05/2022]
Affiliation(s)
- Michelle Miranda-Velez
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th St., Miami, Florida 33199, United States
| | - Golam Sabbir Sarker
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th St., Miami, Florida 33199, United States
| | - Priya Ramisetty
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th St., Miami, Florida 33199, United States
| | - Sandra Geden
- Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 6900 Lake Nona Blvd., Orlando, Florida 32827, United States
| | - Priscila Cristina Bartolomeu Halicki
- Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 6900 Lake Nona Blvd., Orlando, Florida 32827, United States
| | - Thirunavukkarasu Annamalai
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th St., Miami, Florida 33199, United States
| | - Yuk-Ching Tse-Dinh
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th St., Miami, Florida 33199, United States
- Biomolecular Sciences Institute, Florida International University, 11200 SW 8th St., Miami, Florida 33199, United States
| | - Kyle H. Rohde
- Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, 6900 Lake Nona Blvd., Orlando, Florida 32827, United States
| | - Joong Ho Moon
- Department of Chemistry and Biochemistry, Florida International University, 11200 SW 8th St., Miami, Florida 33199, United States
- Biomolecular Sciences Institute, Florida International University, 11200 SW 8th St., Miami, Florida 33199, United States
| |
Collapse
|
|
10
|
Wang Z, Wang S, Jia Z, Zhao Y, Yang M, Yan W, Chen T, Xiang D, Shao R, Liu Y. Establishment and characterization of an immortalized epithelial cell line from human gallbladder. Front Oncol 2022; 12:994087. [PMID: 36387215 PMCID: PMC9650220 DOI: 10.3389/fonc.2022.994087] [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: 07/14/2022] [Accepted: 09/26/2022] [Indexed: 11/25/2022] Open
Abstract
Background Although a plethora of studies have employed multiple gallbladder cancer (GBC) cell lines, it is surprisingly noted that there is still lack of a normal gallbladder epithelial cell line as a normal counterpart, thus impeding substantially the progress of mechanistic studies on the transformation of normal epithelial cells to cancer. Here, we created a normal gallbladder epithelial cell line named L-2F7 from human gallbladder tissue. Methods Gallbladder tissues from a diagnosed cholecystitis female patient were collected, and epithelial cells were enriched by magnetic cell sorting. Then, the cells were immortalized by co-introduction of human telomerase reverse transcriptase (hTERT) and Simian virus 40 large T antigen (LT-SV40) via a lentivirus infection system. After clonal selection and isolation, L-2F7 cells were tested for epithelial markers CK7, CK19, CK20, and CD326, genomic feature, cell proliferation, and migration using Western blot, immunofluorescence, whole genome sequencing, karyotyping, and RNA sequencing. L-2F7 cells were also transplanted to Nude (nu/nu) mice to determine tumorigenicity. Results We successfully identified one single-cell clone named L-2F7 which highly expressed epithelial markers CD326, CK7, CK19, and CK20. This cell line proliferated with a doubling time of 23 h and the epithelial morphology sustained over 30 passages following immortalization. Transient gene transduction of L-2F7 cells led to expression of exogenous GFP and FLAG protein. L-2F7 cells exhibited both distinct non-synonymous mutations from those of gallbladder cancer tissues and differential non-cancerous gene expression patterns similar to normal tissue. Although they displayed unexpected mobility, L-2F7 cells still lacked the ability to develop tumors. Conclusion We developed a non-cancerous gallbladder epithelial cell line, offering a valuable system for the study of gallbladder cancer and other gallbladder-related disorders.
Collapse
Affiliation(s)
- Ziyi Wang
- Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China
- Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China
| | - Shijia Wang
- Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China
- Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China
| | - Ziheng Jia
- Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China
- Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China
| | - Yuhao Zhao
- Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China
- Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China
| | - Mao Yang
- Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China
- Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China
| | - Weikang Yan
- Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China
- Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China
| | - Tao Chen
- Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China
- Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China
| | - Dongxi Xiang
- Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China
- Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China
- *Correspondence: Dongxi Xiang, ; Rong Shao, ; Yingbin Liu,
| | - Rong Shao
- Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China
- Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Department of Pharmacology and Biochemistry, Shanghai Jiao Tong University School of Medicine, Shanghai, China
- *Correspondence: Dongxi Xiang, ; Rong Shao, ; Yingbin Liu,
| | - Yingbin Liu
- Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
- Shanghai Cancer Institute, State Key Laboratory of Oncogenes and Related Genes, Shanghai, China
- Shanghai Key Laboratory of Biliary Tract Disease, Renji Hospital, Shanghai, China
- Shanghai Research Center of Biliary Tract Disease, Renji Hospital, Shanghai, China
- *Correspondence: Dongxi Xiang, ; Rong Shao, ; Yingbin Liu,
| |
Collapse
|
|
11
|
Verdura S, Encinar JA, Fernández-Arroyo S, Joven J, Cuyàs E, Bosch-Barrera J, Menendez JA. Silibinin Suppresses the Hyperlipidemic Effects of the ALK-Tyrosine Kinase Inhibitor Lorlatinib in Hepatic Cells. Int J Mol Sci 2022; 23:9986. [PMID: 36077379 PMCID: PMC9456400 DOI: 10.3390/ijms23179986] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2022] [Revised: 08/27/2022] [Accepted: 08/30/2022] [Indexed: 11/25/2022] Open
Abstract
The third-generation anaplastic lymphoma tyrosine kinase inhibitor (ALK-TKI) lorlatinib has a unique side effect profile that includes hypercholesteremia and hypertriglyceridemia in >80% of lung cancer patients. Here, we tested the hypothesis that lorlatinib might directly promote the accumulation of cholesterol and/or triglycerides in human hepatic cells. We investigated the capacity of the hepatoprotectant silibinin to modify the lipid-modifying activity of lorlatinib. To predict clinically relevant drug−drug interactions if silibinin were used to clinically manage lorlatinib-induced hyperlipidemic effects in hepatic cells, we also explored the capacity of silibinin to interact with and block CYP3A4 activity using in silico computational descriptions and in vitro biochemical assays. A semi-targeted ultrahigh pressure liquid chromatography accurate mass quadrupole time-of-flight mass spectrometry with electrospray ionization (UHPLC-ESI-QTOF-MS/MS)-based lipidomic approach revealed that short-term treatment of hepatic cells with lorlatinib promotes the accumulation of numerous molecular species of cholesteryl esters and triglycerides. Silibinin treatment significantly protected the steady-state lipidome of hepatocytes against the hyperlipidemic actions of lorlatinib. Lipid staining confirmed the ability of lorlatinib to promote neutral lipid overload in hepatocytes upon long-term exposure, which was prevented by co-treatment with silibinin. Computational analyses and cell-free biochemical assays predicted a weak to moderate inhibitory activity of clinically relevant concentrations of silibinin against CYP3A4 when compared with recommended (rosuvastatin) and non-recommended (simvastatin) statins for lorlatinib-associated dyslipidemia. The elevated plasma cholesterol and triglyceride levels in lorlatinib-treated lung cancer patients might involve primary alterations in the hepatic accumulation of lipid intermediates. Silibinin could be clinically explored to reduce the undesirable hyperlipidemic activity of lorlatinib in lung cancer patients.
Collapse
Affiliation(s)
- Sara Verdura
- Metabolism and Cancer Group, Program Against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology, 17007 Girona, Spain
- Girona Biomedical Research Institute (IDIBGI), Salt, 17190 Girona, Spain
| | - José Antonio Encinar
- Institute of Research, Development and Innovation in Biotechnology of Elche (IDiBE) and Molecular and Cell Biology Institute (IBMC), Miguel Hernández University (UMH), 03207 Elche, Spain
| | - Salvador Fernández-Arroyo
- Department of Medicine and Surgery, Universitat Rovira i Virgili, 43204 Reus, Spain
- Unitat de Recerca Biomèdica (URB-CRB), Hospital Universitari de Sant Joan, Institut d’Investigació Sanitaria Pere Virgili, Universitat Rovira i Virgili, 43204 Reus, Spain
| | - Jorge Joven
- Department of Medicine and Surgery, Universitat Rovira i Virgili, 43204 Reus, Spain
- Unitat de Recerca Biomèdica (URB-CRB), Hospital Universitari de Sant Joan, Institut d’Investigació Sanitaria Pere Virgili, Universitat Rovira i Virgili, 43204 Reus, Spain
| | - Elisabet Cuyàs
- Metabolism and Cancer Group, Program Against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology, 17007 Girona, Spain
- Girona Biomedical Research Institute (IDIBGI), Salt, 17190 Girona, Spain
| | - Joaquim Bosch-Barrera
- Girona Biomedical Research Institute (IDIBGI), Salt, 17190 Girona, Spain
- Medical Oncology, Catalan Institute of Oncology, 17007 Girona, Spain
- Department of Medical Sciences, Medical School, University of Girona, 17071 Girona, Spain
| | - Javier A. Menendez
- Metabolism and Cancer Group, Program Against Cancer Therapeutic Resistance (ProCURE), Catalan Institute of Oncology, 17007 Girona, Spain
- Girona Biomedical Research Institute (IDIBGI), Salt, 17190 Girona, Spain
| |
Collapse
|
|
12
|
Three-dimensional (3D) liver cell models - a tool for bridging the gap between animal studies and clinical trials when screening liver accumulation and toxicity of nanobiomaterials. Drug Deliv Transl Res 2022; 12:2048-2074. [PMID: 35507131 PMCID: PMC9066991 DOI: 10.1007/s13346-022-01147-0] [Citation(s) in RCA: 21] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/03/2022] [Indexed: 12/13/2022]
Abstract
Despite the exciting properties and wide-reaching applications of nanobiomaterials (NBMs) in human health and medicine, their translation from bench to bedside is slow, with a predominant issue being liver accumulation and toxicity following systemic administration. In vitro 2D cell-based assays and in vivo testing are the most popular and widely used methods for assessing liver toxicity at pre-clinical stages; however, these fall short in predicting toxicity for NBMs. Focusing on in vitro and in vivo assessment, the accurate prediction of human-specific hepatotoxicity is still a significant challenge to researchers. This review describes the relationship between NBMs and the liver, and the methods for assessing toxicity, focusing on the limitations they bring in the assessment of NBM hepatotoxicity as one of the reasons defining the poor translation for NBMs. We will then present some of the most recent advances towards the development of more biologically relevant in vitro liver methods based on tissue-mimetic 3D cell models and how these could facilitate the translation of NBMs going forward. Finally, we also discuss the low public acceptance and limited uptake of tissue-mimetic 3D models in pre-clinical assessment, despite the demonstrated technical and ethical advantages associated with them.
Collapse
|
|
13
|
Hurrell T, Naidoo J, Scholefield J. Hepatic Models in Precision Medicine: An African Perspective on Pharmacovigilance. Front Genet 2022; 13:864725. [PMID: 35495161 PMCID: PMC9046844 DOI: 10.3389/fgene.2022.864725] [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: 01/28/2022] [Accepted: 03/29/2022] [Indexed: 01/02/2023] Open
Abstract
Pharmaceuticals are indispensable to healthcare as the burgeoning global population is challenged by diseases. The African continent harbors unparalleled genetic diversity, yet remains largely underrepresented in pharmaceutical research and development, which has serious implications for pharmaceuticals approved for use within the African population. Adverse drug reactions (ADRs) are often underpinned by unique variations in genes encoding the enzymes responsible for their uptake, metabolism, and clearance. As an example, individuals of African descent (14-34%) harbor an exclusive genetic variant in the gene encoding a liver metabolizing enzyme (CYP2D6) which reduces the efficacy of the breast cancer chemotherapeutic Tamoxifen. However, CYP2D6 genotyping is not required prior to dispensing Tamoxifen in sub-Saharan Africa. Pharmacogenomics is fundamental to precision medicine and the absence of its implementation suggests that Africa has, to date, been largely excluded from the global narrative around stratified healthcare. Models which could address this need, include primary human hepatocytes, immortalized hepatic cell lines, and induced pluripotent stem cell (iPSC) derived hepatocyte-like cells. Of these, iPSCs, are promising as a functional in vitro model for the empirical evaluation of drug metabolism. The scale with which pharmaceutically relevant African genetic variants can be stratified, the expediency with which these platforms can be established, and their subsequent sustainability suggest that they will have an important role to play in the democratization of stratified healthcare in Africa. Here we discuss the requirement for African hepatic models, and their implications for the future of pharmacovigilance on the African continent.
Collapse
Affiliation(s)
- Tracey Hurrell
- Bioengineering and Integrated Genomics Group, Next Generation Health Cluster, Council for Scientific and Industrial Research, Pretoria, South Africa
| | - Jerolen Naidoo
- Bioengineering and Integrated Genomics Group, Next Generation Health Cluster, Council for Scientific and Industrial Research, Pretoria, South Africa
| | - Janine Scholefield
- Bioengineering and Integrated Genomics Group, Next Generation Health Cluster, Council for Scientific and Industrial Research, Pretoria, South Africa
- Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
| |
Collapse
|
|
14
|
Sharifi Tabar M, Francis H, Yeo D, Bailey CG, Rasko JEJ. Mapping oncogenic protein interactions for precision medicine. Int J Cancer 2022; 151:7-19. [PMID: 35113472 PMCID: PMC9306658 DOI: 10.1002/ijc.33954] [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: 09/27/2021] [Revised: 01/25/2022] [Accepted: 01/26/2022] [Indexed: 11/10/2022]
Abstract
Normal protein‐protein interactions (normPPIs) occur with high fidelity to regulate almost every physiological process. In cancer, this highly organised and precisely regulated network is disrupted, hijacked or reprogrammed resulting in oncogenic protein‐protein interactions (oncoPPIs). OncoPPIs, which can result from genomic alterations, are a hallmark of many types of cancers. Recent technological advances in the field of mass spectrometry (MS)‐based interactomics, structural biology and drug discovery have prompted scientists to identify and characterise oncoPPIs. Disruption of oncoPPI interfaces has become a major focus of drug discovery programs and has resulted in the use of PPI‐specific drugs clinically. However, due to several technical hurdles, studies to build a reference oncoPPI map for various cancer types have not been undertaken. Therefore, there is an urgent need for experimental workflows to overcome the existing challenges in studying oncoPPIs in various cancers and to build comprehensive reference maps. Here, we discuss the important hurdles for characterising oncoPPIs and propose a three‐phase multidisciplinary workflow to identify and characterise oncoPPIs. Systematic identification of cancer‐type‐specific oncogenic interactions will spur new opportunities for PPI‐focused drug discovery projects and precision medicine.
Collapse
Affiliation(s)
- Mehdi Sharifi Tabar
- Gene & Stem Cell Therapy Program Centenary Institute, The University of Sydney, Camperdown, NSW, Australia.,Cancer & Gene Regulation Laboratory Centenary Institute, The University of Sydney, Camperdown, NSW, Australia.,Faculty of Medicine & Health, The University of Sydney, Sydney, NSW, Australia
| | - Habib Francis
- Gene & Stem Cell Therapy Program Centenary Institute, The University of Sydney, Camperdown, NSW, Australia.,Cancer & Gene Regulation Laboratory Centenary Institute, The University of Sydney, Camperdown, NSW, Australia.,Faculty of Medicine & Health, The University of Sydney, Sydney, NSW, Australia
| | - Dannel Yeo
- Faculty of Medicine & Health, The University of Sydney, Sydney, NSW, Australia.,Li Ka Shing Cell & Gene Therapy Program, The University of Sydney, Camperdown, NSW, Australia.,Cell & Molecular Therapies, Royal Prince Alfred Hospital, Sydney Local Health District, Camperdown, NSW, Australia
| | - Charles G Bailey
- Gene & Stem Cell Therapy Program Centenary Institute, The University of Sydney, Camperdown, NSW, Australia.,Cancer & Gene Regulation Laboratory Centenary Institute, The University of Sydney, Camperdown, NSW, Australia.,Faculty of Medicine & Health, The University of Sydney, Sydney, NSW, Australia
| | - John E J Rasko
- Gene & Stem Cell Therapy Program Centenary Institute, The University of Sydney, Camperdown, NSW, Australia.,Faculty of Medicine & Health, The University of Sydney, Sydney, NSW, Australia.,Li Ka Shing Cell & Gene Therapy Program, The University of Sydney, Camperdown, NSW, Australia.,Cell & Molecular Therapies, Royal Prince Alfred Hospital, Sydney Local Health District, Camperdown, NSW, Australia
| |
Collapse
|
|
15
|
Nagul Kumar S, Buvanesvaragurunathan K, Govindaraj R, Rajan S, Balakrishna K, Shirota O, Radha A, Perumal P, Ignacimuthu S. Hepatoprotective constituents from Macrocybe gigantea (Massee) Pegler & Lodge. Int J Med Mushrooms 2022; 24:35-47. [DOI: 10.1615/intjmedmushrooms.2022045329] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/13/2022]
|
|
16
|
Kumar S, Curran JE, Williams-Blangero S, Blangero J. Efficient Generation of Functional Hepatocytes from Human Induced Pluripotent Stem Cells for Disease Modeling and Disease Gene Discovery. Methods Mol Biol 2022; 2549:85-101. [PMID: 33772461 PMCID: PMC11131577 DOI: 10.1007/7651_2021_375] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/01/2023]
Abstract
In vitro hepatocyte cell models are being used to study the pathogenesis of liver disease and in the discovery and preclinical stages of drug development. The culture of hepatic cell lines and primary hepatocytes as in vitro cell models has been carried out for several decades. However, hepatic cell lines (hepatic carcinoma generated or immortalized) have limited accuracy when recapitulating complex physiological functions of the liver. Additionally, primary hepatocytes sourced from human cadavers or medical biopsies are difficult to obtain due to sourcing limitations, particularly for large-scale population studies or in applications requiring large number of cells. Hepatocyte cultures differentiated from human embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) overcome in large part the limitations of traditional hepatocyte in vitro models. In this chapter, we described an efficient protocol routinely used in our laboratory to differentiate human iPSCs into functional hepatocyte cultures for in vitro modeling of liver function and disease. The protocol uses a three-stage differentiation strategy to generate functional hepatocytes from human iPSCs. The differentiated cells show characteristic hepatocyte morphology including flat and polygonal shape, distinct round nuclei, and presence of biliary canaliculi and they express hepatic markers alpha-fetoprotein (AFP), albumin (ALB), E-cadherin (CHD1), hepatocyte nuclear factor 4 alpha (HNF4α), and actin.
Collapse
Affiliation(s)
- Satish Kumar
- Department of Human Genetics and South Texas Diabetes and Obesity Institute, University of Texas Rio Grande Valley School of Medicine, McAllen, TX, USA.
| | - Joanne E Curran
- Department of Human Genetics and South Texas Diabetes and Obesity Institute, University of Texas Rio Grande Valley School of Medicine, Brownsville, TX, USA
| | - Sarah Williams-Blangero
- Department of Human Genetics and South Texas Diabetes and Obesity Institute, University of Texas Rio Grande Valley School of Medicine, McAllen, TX, USA
- Department of Human Genetics and South Texas Diabetes and Obesity Institute, University of Texas Rio Grande Valley School of Medicine, Brownsville, TX, USA
| | - John Blangero
- Department of Human Genetics and South Texas Diabetes and Obesity Institute, University of Texas Rio Grande Valley School of Medicine, Brownsville, TX, USA
| |
Collapse
|
|
17
|
Hadjittofi C, Feretis M, Martin J, Harper S, Huguet E. Liver regeneration biology: Implications for liver tumour therapies. World J Clin Oncol 2021; 12:1101-1156. [PMID: 35070734 PMCID: PMC8716989 DOI: 10.5306/wjco.v12.i12.1101] [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: 04/27/2021] [Revised: 06/22/2021] [Accepted: 11/28/2021] [Indexed: 02/06/2023] Open
Abstract
The liver has remarkable regenerative potential, with the capacity to regenerate after 75% hepatectomy in humans and up to 90% hepatectomy in some rodent models, enabling it to meet the challenge of diverse injury types, including physical trauma, infection, inflammatory processes, direct toxicity, and immunological insults. Current understanding of liver regeneration is based largely on animal research, historically in large animals, and more recently in rodents and zebrafish, which provide powerful genetic manipulation experimental tools. Whilst immensely valuable, these models have limitations in extrapolation to the human situation. In vitro models have evolved from 2-dimensional culture to complex 3 dimensional organoids, but also have shortcomings in replicating the complex hepatic micro-anatomical and physiological milieu. The process of liver regeneration is only partially understood and characterized by layers of complexity. Liver regeneration is triggered and controlled by a multitude of mitogens acting in autocrine, paracrine, and endocrine ways, with much redundancy and cross-talk between biochemical pathways. The regenerative response is variable, involving both hypertrophy and true proliferative hyperplasia, which is itself variable, including both cellular phenotypic fidelity and cellular trans-differentiation, according to the type of injury. Complex interactions occur between parenchymal and non-parenchymal cells, and regeneration is affected by the status of the liver parenchyma, with differences between healthy and diseased liver. Finally, the process of termination of liver regeneration is even less well understood than its triggers. The complexity of liver regeneration biology combined with limited understanding has restricted specific clinical interventions to enhance liver regeneration. Moreover, manipulating the fundamental biochemical pathways involved would require cautious assessment, for fear of unintended consequences. Nevertheless, current knowledge provides guiding principles for strategies to optimise liver regeneration potential.
Collapse
Affiliation(s)
- Christopher Hadjittofi
- University Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Center, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Michael Feretis
- University Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Center, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Jack Martin
- University Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Center, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Simon Harper
- University Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Center, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| | - Emmanuel Huguet
- University Department of Surgery, Addenbrookes Hospital, NIHR Comprehensive Biomedical Research and Academic Health Sciences Center, Cambridge University Hospitals NHS Foundation Trust, Cambridge CB2 0QQ, United Kingdom
| |
Collapse
|
|
18
|
Romualdo GR, Leroy K, Costa CJS, Prata GB, Vanderborght B, da Silva TC, Barbisan LF, Andraus W, Devisscher L, Câmara NOS, Vinken M, Cogliati B. In Vivo and In Vitro Models of Hepatocellular Carcinoma: Current Strategies for Translational Modeling. Cancers (Basel) 2021; 13:5583. [PMID: 34771745 PMCID: PMC8582701 DOI: 10.3390/cancers13215583] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/13/2021] [Revised: 11/02/2021] [Accepted: 11/04/2021] [Indexed: 12/24/2022] Open
Abstract
Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer-related death globally. HCC is a complex multistep disease and usually emerges in the setting of chronic liver diseases. The molecular pathogenesis of HCC varies according to the etiology, mainly caused by chronic hepatitis B and C virus infections, chronic alcohol consumption, aflatoxin-contaminated food, and non-alcoholic fatty liver disease associated with metabolic syndrome or diabetes mellitus. The establishment of HCC models has become essential for both basic and translational research to improve our understanding of the pathophysiology and unravel new molecular drivers of this disease. The ideal model should recapitulate key events observed during hepatocarcinogenesis and HCC progression in view of establishing effective diagnostic and therapeutic strategies to be translated into clinical practice. Despite considerable efforts currently devoted to liver cancer research, only a few anti-HCC drugs are available, and patient prognosis and survival are still poor. The present paper provides a state-of-the-art overview of in vivo and in vitro models used for translational modeling of HCC with a specific focus on their key molecular hallmarks.
Collapse
Affiliation(s)
- Guilherme Ribeiro Romualdo
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), São Paulo 05508-270, Brazil; (G.R.R.); (C.J.S.C.); (T.C.d.S.)
- Department of Structural and Functional Biology, Biosciences Institute, São Paulo State University (UNESP), Botucatu 18618-689, Brazil; (G.B.P.); (L.F.B.)
- Department of Pathology, Botucatu Medical School, São Paulo State University (UNESP), Botucatu 18618-687, Brazil
| | - Kaat Leroy
- Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (K.L.); (M.V.)
| | - Cícero Júlio Silva Costa
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), São Paulo 05508-270, Brazil; (G.R.R.); (C.J.S.C.); (T.C.d.S.)
| | - Gabriel Bacil Prata
- Department of Structural and Functional Biology, Biosciences Institute, São Paulo State University (UNESP), Botucatu 18618-689, Brazil; (G.B.P.); (L.F.B.)
- Department of Pathology, Botucatu Medical School, São Paulo State University (UNESP), Botucatu 18618-687, Brazil
| | - Bart Vanderborght
- Gut-Liver Immunopharmacology Unit, Basic and Applied Medical Sciences, Liver Research Center Ghent, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium;
- Hepatology Research Unit, Internal Medicine and Paediatrics, Liver Research Center Ghent, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium;
| | - Tereza Cristina da Silva
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), São Paulo 05508-270, Brazil; (G.R.R.); (C.J.S.C.); (T.C.d.S.)
| | - Luís Fernando Barbisan
- Department of Structural and Functional Biology, Biosciences Institute, São Paulo State University (UNESP), Botucatu 18618-689, Brazil; (G.B.P.); (L.F.B.)
| | - Wellington Andraus
- Department of Gastroenterology, Clinics Hospital, School of Medicine, University of São Paulo (HC-FMUSP), São Paulo 05403-000, Brazil;
| | - Lindsey Devisscher
- Hepatology Research Unit, Internal Medicine and Paediatrics, Liver Research Center Ghent, Faculty of Medicine and Health Sciences, Ghent University, 9000 Ghent, Belgium;
| | - Niels Olsen Saraiva Câmara
- Department of Immunology, Institute of Biomedical Sciences IV, University of São Paulo (USP), São Paulo 05508-000, Brazil;
| | - Mathieu Vinken
- Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, 1090 Brussels, Belgium; (K.L.); (M.V.)
| | - Bruno Cogliati
- Department of Pathology, School of Veterinary Medicine and Animal Science, University of São Paulo (USP), São Paulo 05508-270, Brazil; (G.R.R.); (C.J.S.C.); (T.C.d.S.)
| |
Collapse
|
|
19
|
Pluta KD, Ciezkowska M, Wisniewska M, Wencel A, Pijanowska DG. Cell-based clinical and experimental methods for assisting the function of impaired livers – Present and future of liver support systems. Biocybern Biomed Eng 2021. [DOI: 10.1016/j.bbe.2021.06.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
|
|
20
|
Xu Q. Human Three-Dimensional Hepatic Models: Cell Type Variety and Corresponding Applications. Front Bioeng Biotechnol 2021; 9:730008. [PMID: 34631680 PMCID: PMC8497968 DOI: 10.3389/fbioe.2021.730008] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2021] [Accepted: 08/30/2021] [Indexed: 12/23/2022] Open
Abstract
Owing to retained hepatic phenotypes and functions, human three-dimensional (3D) hepatic models established with diverse hepatic cell types are thought to recoup the gaps in drug development and disease modeling limited by a conventional two-dimensional (2D) cell culture system and species-specific variability in drug metabolizing enzymes and transporters. Primary human hepatocytes, human hepatic cancer cell lines, and human stem cell-derived hepatocyte-like cells are three main hepatic cell types used in current models and exhibit divergent hepatic phenotypes. Primary human hepatocytes derived from healthy hepatic parenchyma resemble in vivo-like genetic and metabolic profiling. Human hepatic cancer cell lines are unlimitedly reproducible and tumorigenic. Stem cell-derived hepatocyte-like cells derived from patients are promising to retain the donor's genetic background. It has been suggested in some studies that unique properties of cell types endue them with benefits in different research fields of in vitro 3D modeling paradigm. For instance, the primary human hepatocyte was thought to be the gold standard for hepatotoxicity study, and stem cell-derived hepatocyte-like cells have taken a main role in personalized medicine and regenerative medicine. However, the comprehensive review focuses on the hepatic cell type variety, and corresponding applications in 3D models are sparse. Therefore, this review summarizes the characteristics of different cell types and discusses opportunities of different cell types in drug development, liver disease modeling, and liver transplantation.
Collapse
Affiliation(s)
- Qianqian Xu
- School of Chinese Medicine, and Centre for Cancer and Inflammation Research, Hong Kong Baptist University, Hong Kong, China
| |
Collapse
|
|
21
|
Host bioenergetic parameters reveal cytotoxicity of anti-tuberculosis drugs undetected using conventional viability assays. Antimicrob Agents Chemother 2021; 65:e0093221. [PMID: 34339269 PMCID: PMC8448146 DOI: 10.1128/aac.00932-21] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/30/2022] Open
Abstract
High attrition rates in tuberculosis (TB) drug development have been largely attributed to safety, which is likely due to the use of endpoint assays measuring cell viability to detect drug cytotoxicity. In drug development for cancer, metabolic, and neurological disorders and for antibiotics, cytotoxicity is increasingly being assessed using extracellular flux (XF) analysis, which measures cellular bioenergetic metabolism in real time. Here, we adopt the XF platform to investigate the cytotoxicity of drugs currently used in TB treatment on the bioenergetic metabolism of HepG2 cells, THP-1 macrophages, and human monocyte-derived macrophages (hMDMs). We found that the XF analysis reveals earlier drug-induced effects on the cells’ bioenergetic metabolism prior to cell death, measured by conventional viability assays. Furthermore, each cell type has a distinct response to drug treatment, suggesting that more than one cell type should be considered to examine cytotoxicity in TB drug development. Interestingly, chemically unrelated drugs with different modes of action on Mycobacterium tuberculosis have similar effects on the bioenergetic parameters of the cells, thus discouraging the prediction of potential cytotoxicity based on chemical structure and mode of action of new chemical entities. The clustering of the drug-induced effects on the hMDM bioenergetic parameters are reflected in the clustering of the effects of the drugs on cytokine production in hMDMs, demonstrating concurrence between the effects of the drugs on the metabolism and functioning of the macrophages. These findings can be used as a benchmark to establish XF analysis as a new tool to assay cytotoxicity in TB drug development.
Collapse
|
|
22
|
Khoo J, Hagemeyer CE, Henstridge DC, Kumble S, Wang TY, Xu R, Gani L, King T, Soh SB, Puar T, Au V, Tan E, Tay TL, Kam C, Teo EK. Effects of water stably-enriched with oxygen as a novel method of tissue oxygenation on mitochondrial function, and as adjuvant therapy for type 2 diabetes in a randomized placebo-controlled trial. PLoS One 2021; 16:e0254619. [PMID: 34260650 PMCID: PMC8279347 DOI: 10.1371/journal.pone.0254619] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2020] [Accepted: 06/07/2021] [Indexed: 01/17/2023] Open
Abstract
BACKGROUND Diabetes mellitus is associated with inadequate delivery of oxygen to tissues. Cellular hypoxia is associated with mitochondrial dysfunction which increases oxidative stress and hyperglycaemia. Hyperbaric oxygenation therapy, which was shown to improve insulin sensitivity, is impractical for regular use. We evaluated the effects of water which is stably-enriched with oxygen (ELO water) to increase arterial blood oxygen levels, on mitochondrial function in the presence of normal- or high-glucose environments, and as glucose-lowering therapy in humans. METHODS We compared arterial blood oxygen levels in Sprague-Dawley rats after 7 days of ad libitum ELO or tap water consumption. Mitochondrial stress testing, and flow cytometry analysis of mitochondrial mass and membrane potential, were performed on human HepG2 cells cultured in four Dulbecco's Modified Eagle Medium media, made with ELO water or regular (control) water, at normal (5.5 mM) or high (25 mM) glucose concentrations. We also randomized 150 adults with type 2 diabetes (mean age 53 years, glycated haemoglobin HbA1c 8.9% [74 mmol/mol], average duration of diabetes 12 years) to drink 1.5 litres daily of bottled ELO water or drinking water. RESULTS ELO water raised arterial oxygen tension pO2 significantly (335 ± 26 vs. 188 ± 18 mmHg, p = 0.006) compared with tap water. In cells cultured in control water, mitochondrial mass and membrane potential were both significantly lower at 25 mM glucose compared with 5.5 mM glucose; in contrast, mitochondrial mass and membrane potential did not differ significantly at normal or high glucose concentrations in cells cultured in ELO water. The high-glucose environment induced a greater mitochondrial proton leak in cells cultured in ELO water compared to cells cultured in control medium at similar glucose concentration. In type 2 diabetic adults, HbA1c decreased significantly (p = 0.002) by 0.3 ± 0.7% (4 ± 8 mmol/mol), with ELO water after 12 weeks of treatment but was unchanged with placebo. CONCLUSIONS ELO water raises arterial blood oxygen levels, appears to have a protective effect on hyperglycaemia-induced reduction in mitochondrial mass and mitochondrial dysfunction, and may be effective adjuvant therapy for type 2 diabetes.
Collapse
Affiliation(s)
- Joan Khoo
- Department of Medicine, Changi General Hospital, Singapore, Singapore
| | | | - Darren C. Henstridge
- Baker Heart and Diabetes Institute, Melbourne, Victoria, Australia
- School of Health Sciences, University of Tasmania, Launceston, Tasmania, Australia
| | - Sumukh Kumble
- NanoBiotechnology Laboratory, Monash University, Melbourne, Victoria, Australia
| | - Ting-Yi Wang
- NanoBiotechnology Laboratory, Monash University, Melbourne, Victoria, Australia
| | - Rong Xu
- NanoBiotechnology Laboratory, Monash University, Melbourne, Victoria, Australia
| | - Linsey Gani
- Department of Medicine, Changi General Hospital, Singapore, Singapore
| | - Thomas King
- Department of Medicine, Changi General Hospital, Singapore, Singapore
| | - Shui-Boon Soh
- Department of Medicine, Changi General Hospital, Singapore, Singapore
| | - Troy Puar
- Department of Medicine, Changi General Hospital, Singapore, Singapore
| | - Vanessa Au
- Department of Medicine, Changi General Hospital, Singapore, Singapore
| | - Eberta Tan
- Department of Medicine, Changi General Hospital, Singapore, Singapore
| | - Tunn-Lin Tay
- Department of Medicine, Changi General Hospital, Singapore, Singapore
| | - Carmen Kam
- Clinical Trials and Research Unit, Changi General Hospital, Singapore, Singapore
| | - Eng-Kiong Teo
- Department of Medicine, Changi General Hospital, Singapore, Singapore
| |
Collapse
|
|
23
|
Siddiqui S, Upadhyay S, Ahmad I, Hussain A, Ahamed M. Cytotoxicity of Moringa oleifera fruits on human liver cancer and molecular docking analysis of bioactive constituents against caspase-3 enzyme. J Food Biochem 2021; 45:e13720. [PMID: 33856706 DOI: 10.1111/jfbc.13720] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/30/2020] [Revised: 02/18/2021] [Accepted: 03/10/2021] [Indexed: 12/18/2022]
Abstract
Moringa oleifera is an excellent source of phenolics and flavonoids comprise various pharmacological activities. The fourth widespread leading cause of the patients' death is liver cancer. This study was formulated to perform the antiproliferative activity of Moringa oleifera fruit (MOF) extract on human liver cancer HepG2 cells and computational validation of cell death. HepG2 cell line was treated with 25, 50, 75, 100, and 200 µg/ml of MOF extract for 48 hr, and antiproliferative activity was analyzed using MTT assay, nuclear condensation, annexin V-FITC/PI double stain, ROS generation, and apoptosis executioner enzyme caspase-3. MOF extract reduced the cell viability significantly (p ˂ .05) by increasing cellular apoptosis which was confirmed by annexin V-FITC/PI staining assay. In addition, MOF stimulated intracellular ROS production and subsequently induced caspase-3 activity depending upon dose. In silico analysis revealed the good binding interaction between amino acid residues of caspase-3 (PDB ID: 1GFW) protein and selected active constituents of MOF. PASS analyses of the phytoconstituents showed no violation of Lipinski's rule of five. Analysis of drug-likeness and toxicity measurement exhibited drug-like candidates with no predicted toxicity. In conclusion, this study showed the potential anticancer activity of MOF extract which may be valuable source for anticancer drug development. PRACTICAL APPLICATIONS: Moringa oleifera fruit extract induced the anti-proliferative activity against human hepatocellular carcinoma HepG2 cells through ROS-mediated apoptosis and activation of caspase-3 enzyme. Structure-based virtual screening study between bioactive components of Moringa oleifera fruits and apoptosis executioner caspase-3 enzyme has validated the anti-proliferative activity of Moringa oleifera fruit extract. Interestingly, active phytoconstituents of Moringa oleifera fruit exhibited drug-like candidates with no predicted toxicity. Thus, Moringa oleifera fruit could be used as valuable source for anticancer drug development against human liver cancer with relatively non-toxic to healthy cells.
Collapse
Affiliation(s)
- Sahabjada Siddiqui
- Department of Biotechnology, Era's Lucknow Medical College & Hospital, Era University, Lucknow, India
| | - Shivbrat Upadhyay
- Department of Biotechnology, Era's Lucknow Medical College & Hospital, Era University, Lucknow, India
| | - Imran Ahmad
- Department of Biochemistry, King George's Medical University, Lucknow, India
| | | | - Maqusood Ahamed
- King Abdullah Institute for Nanotechnology, King Saud University, Riyadh, Saudi Arabia
| |
Collapse
|
|
24
|
|
|
25
|
Sakolish C, Reese CE, Luo YS, Valdiviezo A, Schurdak ME, Gough A, Taylor DL, Chiu WA, Vernetti LA, Rusyn I. Analysis of reproducibility and robustness of a human microfluidic four-cell liver acinus microphysiology system (LAMPS). Toxicology 2021; 448:152651. [PMID: 33307106 PMCID: PMC7785655 DOI: 10.1016/j.tox.2020.152651] [Citation(s) in RCA: 23] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2020] [Revised: 10/06/2020] [Accepted: 11/27/2020] [Indexed: 02/07/2023]
Abstract
A human microfluidic four-cell liver acinus microphysiology system (LAMPS), was evaluated for reproducibility and robustness as a model for drug pharmacokinetics and toxicology. The model was constructed using primary human hepatocytes or human induced pluripotent stem cell (iPSC)-derived hepatocytes and 3 human cell lines for the endothelial, Kupffer and stellate cells. The model was tested in two laboratories and demonstrated to be reproducible in terms of basal function of hepatocytes, Terfenadine metabolism, and effects of Tolcapone (88 μM), Troglitazone (150 μM), and caffeine (600 μM) over 9 days in culture. Additional experiments compared basal outputs of albumin, urea, lactate dehydrogenase (LDH) and tumor necrosis factor (TNF)α, as well as drug metabolism and toxicity in the LAMPS model, and in 2D cultures seeded with either primary hepatocytes or iPSC-hepatocytes. Further experiments to study the effects of Terfenadine (10 μM), Tolcapone (88 μM), Trovafloxacin (150 μM with or without 1 μg/mL lipopolysaccharide), Troglitazone (28 μM), Rosiglitazone (0.8 μM), Pioglitazone (3 μM), and caffeine (600 μM) were carried out over 10 days. We found that both primary human hepatocytes and iPSC-derived hepatocytes in 3D culture maintained excellent basal liver function and Terfenadine metabolism over 10 days compared the same cells in 2D cultures. In 2D, non-overlay monolayer cultures, both cell types lost hepatocyte phenotypes after 48 h. With respect to drug effects, both cell types demonstrated comparable and more human-relevant effects in LAMPS, as compared to 2D cultures. Overall, these studies show that LAMPS is a robust and reproducible in vitro liver model, comparable in performance when seeded with either primary human hepatocytes or iPSC-derived hepatocytes, and more physiologically and clinically relevant than 2D monolayer cultures.
Collapse
Affiliation(s)
- Courtney Sakolish
- Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843, USA
| | - Celeste E Reese
- Drug Discovery Institute and Department of Computational & Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
| | - Yu-Syuan Luo
- Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843, USA
| | - Alan Valdiviezo
- Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843, USA
| | - Mark E Schurdak
- Drug Discovery Institute and Department of Computational & Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
| | - Albert Gough
- Drug Discovery Institute and Department of Computational & Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
| | - D Lansing Taylor
- Drug Discovery Institute and Department of Computational & Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
| | - Weihsueh A Chiu
- Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843, USA
| | - Lawrence A Vernetti
- Drug Discovery Institute and Department of Computational & Systems Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
| | - Ivan Rusyn
- Department of Veterinary Integrative Biosciences, Texas A&M University, College Station, TX 77843, USA.
| |
Collapse
|
|
26
|
Soret PA, Magusto J, Housset C, Gautheron J. In Vitro and In Vivo Models of Non-Alcoholic Fatty Liver Disease: A Critical Appraisal. J Clin Med 2020; 10:jcm10010036. [PMID: 33374435 PMCID: PMC7794936 DOI: 10.3390/jcm10010036] [Citation(s) in RCA: 80] [Impact Index Per Article: 16.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2020] [Revised: 12/22/2020] [Accepted: 12/23/2020] [Indexed: 02/07/2023] Open
Abstract
Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), represents the hepatic manifestation of obesity and metabolic syndrome. Due to the spread of the obesity epidemic, NAFLD is becoming the most common chronic liver disease and one of the principal indications for liver transplantation. However, no pharmacological treatment is currently approved to prevent the outbreak of NASH, which leads to fibrosis and cirrhosis. Preclinical research is required to improve our knowledge of NAFLD physiopathology and to identify new therapeutic targets. In the present review, we summarize advances in NAFLD preclinical models from cellular models, including new bioengineered platforms, to in vivo models, with a particular focus on genetic and dietary mouse models. We aim to discuss the advantages and limits of these different models.
Collapse
Affiliation(s)
- Pierre-Antoine Soret
- Centre de Recherche Saint-Antoine (CRSA), Sorbonne Université, Inserm, 75012 Paris, France; (P.-A.S.); (J.M.); (C.H.)
- Assistance Publique-Hôpitaux de Paris (AP-HP), Hepatology Department, Reference Center for Inflammatory Biliary Diseases and Autoimmune Hepatitis, Saint-Antoine Hospital, 75012 Paris, France
| | - Julie Magusto
- Centre de Recherche Saint-Antoine (CRSA), Sorbonne Université, Inserm, 75012 Paris, France; (P.-A.S.); (J.M.); (C.H.)
- Institute of Cardiometabolism and Nutrition (ICAN), Sorbonne Université, Inserm, AP-HP, 75013 Paris, France
| | - Chantal Housset
- Centre de Recherche Saint-Antoine (CRSA), Sorbonne Université, Inserm, 75012 Paris, France; (P.-A.S.); (J.M.); (C.H.)
- Assistance Publique-Hôpitaux de Paris (AP-HP), Hepatology Department, Reference Center for Inflammatory Biliary Diseases and Autoimmune Hepatitis, Saint-Antoine Hospital, 75012 Paris, France
- Institute of Cardiometabolism and Nutrition (ICAN), Sorbonne Université, Inserm, AP-HP, 75013 Paris, France
| | - Jérémie Gautheron
- Centre de Recherche Saint-Antoine (CRSA), Sorbonne Université, Inserm, 75012 Paris, France; (P.-A.S.); (J.M.); (C.H.)
- Institute of Cardiometabolism and Nutrition (ICAN), Sorbonne Université, Inserm, AP-HP, 75013 Paris, France
- Correspondence:
| |
Collapse
|
|
27
|
Cox CR, Lynch S, Goldring C, Sharma P. Current Perspective: 3D Spheroid Models Utilizing Human-Based Cells for Investigating Metabolism-Dependent Drug-Induced Liver Injury. FRONTIERS IN MEDICAL TECHNOLOGY 2020; 2:611913. [PMID: 35047893 PMCID: PMC8757888 DOI: 10.3389/fmedt.2020.611913] [Citation(s) in RCA: 34] [Impact Index Per Article: 6.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2020] [Accepted: 11/04/2020] [Indexed: 12/21/2022] Open
Abstract
Drug-induced liver injury (DILI) remains a leading cause for the withdrawal of approved drugs. This has significant financial implications for pharmaceutical companies, places increasing strain on global health services, and causes harm to patients. For these reasons, it is essential that in-vitro liver models are capable of detecting DILI-positive compounds and their underlying mechanisms, prior to their approval and administration to patients or volunteers in clinical trials. Metabolism-dependent DILI is an important mechanism of drug-induced toxicity, which often involves the CYP450 family of enzymes, and is associated with the production of a chemically reactive metabolite and/or inefficient removal and accumulation of potentially toxic compounds. Unfortunately, many of the traditional in-vitro liver models fall short of their in-vivo counterparts, failing to recapitulate the mature hepatocyte phenotype, becoming metabolically incompetent, and lacking the longevity to investigate and detect metabolism-dependent DILI and those associated with chronic and repeat dosing regimens. Nevertheless, evidence is gathering to indicate that growing cells in 3D formats can increase the complexity of these models, promoting a more mature-hepatocyte phenotype and increasing their longevity, in vitro. This review will discuss the use of 3D in vitro models, namely spheroids, organoids, and perfusion-based systems to establish suitable liver models to investigate metabolism-dependent DILI.
Collapse
Affiliation(s)
- Christopher R. Cox
- Department of Pharmacology and Experimental Therapeutics, MRC Centre for Drug Safety Science, Institute of Systems, Molecular & Integrative Biology, University of Liverpool, Liverpool, United Kingdom
- Department of Cardiovascular and Metabolic Medicine, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, United Kingdom
- *Correspondence: Christopher R. Cox
| | - Stephen Lynch
- Department of Pharmacology and Experimental Therapeutics, MRC Centre for Drug Safety Science, Institute of Systems, Molecular & Integrative Biology, University of Liverpool, Liverpool, United Kingdom
| | - Christopher Goldring
- Department of Pharmacology and Experimental Therapeutics, MRC Centre for Drug Safety Science, Institute of Systems, Molecular & Integrative Biology, University of Liverpool, Liverpool, United Kingdom
| | - Parveen Sharma
- Department of Pharmacology and Experimental Therapeutics, MRC Centre for Drug Safety Science, Institute of Systems, Molecular & Integrative Biology, University of Liverpool, Liverpool, United Kingdom
- Department of Cardiovascular and Metabolic Medicine, Institute of Life Course and Medical Sciences, University of Liverpool, Liverpool, United Kingdom
- Liverpool Centre for Cardiovascular Science, Liverpool, United Kingdom
| |
Collapse
|
|
28
|
Panoutsopoulos AA. Organoids, Assembloids, and Novel Biotechnology: Steps Forward in Developmental and Disease-Related Neuroscience. Neuroscientist 2020; 27:463-472. [PMID: 32981451 DOI: 10.1177/1073858420960112] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
In neuroscience research, the efforts to find the model through which we can mimic the in vivo microenvironment of a developing or defective brain have been everlasting. While model organisms are used for over a hundred years, many more methods have been introduced with immortalized or primary cell lines and later induced pluripotent stem cells and organoids to be some of these. As the use of organoids becomes more and more common by many laboratories in biology and neuroscience in particular, it is crucial to deeper understand the challenges and possible pitfalls of their application in research, many of which can be surpassed with the support of state-of-the art bioengineering solutions. In this review, after a brief chronicle of the path to the discovery of organoids, we focus on the latest approaches to study neuroscience related topics with organoids, such as the use of assembloids, CRISPR technology, patch-clamp and optogenetics techniques and discuss how modern 3-dimensional biomaterials, miniaturized bioreactors and microfluidic chips can help to overcome the disadvantages of their use.
Collapse
Affiliation(s)
- Alexios A Panoutsopoulos
- Department of Pathology and Laboratory Medicine, University of California, Davis, Sacramento, CA, USA.,Institute for Pediatric Regenerative Medicine, Shriners Hospitals for Children-Northern California, Sacramento, CA, USA
| |
Collapse
|
|
29
|
Zidarič T, Milojević M, Vajda J, Vihar B, Maver U. Cultured Meat: Meat Industry Hand in Hand with Biomedical Production Methods. FOOD ENGINEERING REVIEWS 2020. [DOI: 10.1007/s12393-020-09253-w] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
|
|
30
|
Kaczor-Kamińska M, Stalińska K, Kamiński K, Pisarek A, Maziarz U, Feldman A, Wróbel M. Murine cellular model of mucopolysaccharidosis, type IIIB (MPS IIIB) - A preliminary study with particular emphasis on the non-oxidative l-cysteine metabolism. Biochimie 2020; 174:84-94. [PMID: 32335228 DOI: 10.1016/j.biochi.2020.04.015] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/15/2020] [Revised: 04/12/2020] [Accepted: 04/14/2020] [Indexed: 01/22/2023]
Abstract
The lack of the N-alpha-glucosaminidase (Naglu) is responsible for the incidence of a rare disease - mucopolysaccharidosis, type IIIB (MPS IIIB). To date, studies have been conducted based on cells derived from patients suffering from MPS or using in vivo MPS mouse models. These limitations have allowed for defining our research goal - to create and characterize the first in vitro murine cellular MPS IIIB model. In the current work we present a new, stable cell line with confirmed accumulation of glycosaminoglycans. The line stability was achieved by immortalization using a lentivirus carrying the T-antigens of SV40. The Naglu-/- cells were confirmed to produce no Naglu enzyme. To confirm the proper functioning of the in vitro MPS IIIB model, we determined the activity and expression of cystathionine γ-lyase, rhodanese and 3-mercaptopyruvate sulfurtransferase, as well as the level of low molecular-weight thiols (reduced and oxidized glutathione, cysteine and cystine). The results were referred to our earlier findings originating from the studies on the tissues of the Naglu-/- mice that were used to create the lines. The results obtained in the Naglu-/- cells were in accordance with the results found in the mouse model of MPS IIIB. It suggests that the presented murine Naglu-/- cell lines might be a convenient in vitro model of MPS IIIB.
Collapse
Affiliation(s)
- Marta Kaczor-Kamińska
- Jagiellonian University, Medical College, Faculty of Medicine, Chair of Medical Biochemistry, Kopernika 7 St., 31-034, Krakow, Poland
| | - Krystyna Stalińska
- Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Gronostajowa 7 St., 30-387, Krakow, Poland
| | - Kamil Kamiński
- Jagiellonian University, Faculty of Chemistry, Gronostajowa 2 St., 30-387, Krakow, Poland.
| | - Aleksandra Pisarek
- Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Gronostajowa 7 St., 30-387, Krakow, Poland
| | - Urszula Maziarz
- Jagiellonian University, Faculty of Chemistry, Gronostajowa 2 St., 30-387, Krakow, Poland
| | - Arleta Feldman
- Team Sanfilippo Foundation, Ronkonkoma, NY, 11779, United States
| | - Maria Wróbel
- Jagiellonian University, Medical College, Faculty of Medicine, Chair of Medical Biochemistry, Kopernika 7 St., 31-034, Krakow, Poland
| |
Collapse
|
|
31
|
van Gorp PRR, Trines SA, Pijnappels DA, de Vries AAF. Multicellular In vitro Models of Cardiac Arrhythmias: Focus on Atrial Fibrillation. Front Cardiovasc Med 2020; 7:43. [PMID: 32296716 PMCID: PMC7138102 DOI: 10.3389/fcvm.2020.00043] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2019] [Accepted: 03/06/2020] [Indexed: 12/13/2022] Open
Abstract
Atrial fibrillation (AF) is the most common cardiac arrhythmia in clinical practice with a large socioeconomic impact due to its associated morbidity, mortality, reduction in quality of life and health care costs. Currently, antiarrhythmic drug therapy is the first line of treatment for most symptomatic AF patients, despite its limited efficacy, the risk of inducing potentially life-threating ventricular tachyarrhythmias as well as other side effects. Alternative, in-hospital treatment modalities consisting of electrical cardioversion and invasive catheter ablation improve patients' symptoms, but often have to be repeated and are still associated with serious complications and only suitable for specific subgroups of AF patients. The development and progression of AF generally results from the interplay of multiple disease pathways and is accompanied by structural and functional (e.g., electrical) tissue remodeling. Rational development of novel treatment modalities for AF, with its many different etiologies, requires a comprehensive insight into the complex pathophysiological mechanisms. Monolayers of atrial cells represent a simplified surrogate of atrial tissue well-suited to investigate atrial arrhythmia mechanisms, since they can easily be used in a standardized, systematic and controllable manner to study the role of specific pathways and processes in the genesis, perpetuation and termination of atrial arrhythmias. In this review, we provide an overview of the currently available two- and three-dimensional multicellular in vitro systems for investigating the initiation, maintenance and termination of atrial arrhythmias and AF. This encompasses cultures of primary (animal-derived) atrial cardiomyocytes (CMs), pluripotent stem cell-derived atrial-like CMs and (conditionally) immortalized atrial CMs. The strengths and weaknesses of each of these model systems for studying atrial arrhythmias will be discussed as well as their implications for future studies.
Collapse
Affiliation(s)
| | | | | | - Antoine A. F. de Vries
- Laboratory of Experimental Cardiology, Department of Cardiology, Leiden University Medical Center, Leiden, Netherlands
| |
Collapse
|
|
32
|
Ishikawa T, Suwanai H, Shikuma J, Suzuki R, Yamanaka T, Odawara M, Inazu M. Protein kinase C promotes choline transporter‑like protein 1 function via improved cell surface expression in immortalized human hepatic cells. Mol Med Rep 2019; 21:777-785. [PMID: 31974614 PMCID: PMC6947888 DOI: 10.3892/mmr.2019.10894] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/06/2019] [Accepted: 10/16/2019] [Indexed: 12/30/2022] Open
Abstract
Choline is used to synthesize phospholipids and a lack of choline induces a number of liver-related diseases, including non-alcoholic steatohepatitis. The current study characterized the choline uptake system, at molecular and functional levels, in the immortalized human hepatic cell line, Fa2N-4, to identify the specific choline transporter involved in choline uptake. The present study also assesed whether choline deficiency or the inhibited choline uptake affected cell viability and apoptosis. Reverse transcription-quantitative polymerase chain reaction (PCR) revealed choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein expression in Fa2N-4 cells. [Methyl-3H]choline studies revealed choline uptake was saturable and mediated by a single transport system that functioned in a Na+-independent but pH-dependent manner, which was similar to CTL1. Hemicholinium-3 (HC-3), which is a choline uptake inhibitor, and choline deficiency inhibited cell viability, increased caspase-3 and −7 activities, and increased fluorescein isothiocyanate-Annexin V immunofluorescent staining indicated apoptosis. Immunofluorescent staining also revealed CTL1 and CTL2 localized in plasma and mitochondrial membranes, respectively. [Methyl-3H]choline uptake was enhanced by a protein kinase C (PKC) activator, phorbol-12-myristate 13-acetate (PMA). Immunofluorescence staining and western blot analysis demonstrated increased CTL1 expression on the cell membrane following PMA treatment. The results of current study indicated that extracellular choline is primarily transported via CTL1, relying on a direct H+ gradient that functions as a driving force in Fa2N-4 cells. Furthermore, it was hypothesized that CTL1 and the choline uptake system are strongly associated with cell survival, and that the choline uptake system is modulated by PKC signaling via increased CTL1 expression on the cell surface. These findings provide further insights into the pathogenesis of liver disease involving choline metabolism.
Collapse
Affiliation(s)
- Takuya Ishikawa
- Department of Diabetes, Metabolism and Endocrinology, Tokyo Medical University, Tokyo 160‑0023, Japan
| | - Hirotsugu Suwanai
- Department of Diabetes, Metabolism and Endocrinology, Tokyo Medical University, Tokyo 160‑0023, Japan
| | - Junpei Shikuma
- Department of Diabetes, Metabolism and Endocrinology, Tokyo Medical University, Tokyo 160‑0023, Japan
| | - Ryo Suzuki
- Department of Diabetes, Metabolism and Endocrinology, Tokyo Medical University, Tokyo 160‑0023, Japan
| | - Tsuyoshi Yamanaka
- Department of Molecular Preventive Medicine, Tokyo Medical University, Tokyo 160‑8402, Japan
| | - Masato Odawara
- Department of Diabetes, Metabolism and Endocrinology, Tokyo Medical University, Tokyo 160‑0023, Japan
| | - Masato Inazu
- Department of Molecular Preventive Medicine, Tokyo Medical University, Tokyo 160‑8402, Japan
| |
Collapse
|
|
33
|
Park E, Kim HK, Jee J, Hahn S, Jeong S, Yoo J. Development of organoid-based drug metabolism model. Toxicol Appl Pharmacol 2019; 385:114790. [DOI: 10.1016/j.taap.2019.114790] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2019] [Revised: 08/27/2019] [Accepted: 10/22/2019] [Indexed: 12/23/2022]
|
|
34
|
Lankatillake C, Huynh T, Dias DA. Understanding glycaemic control and current approaches for screening antidiabetic natural products from evidence-based medicinal plants. PLANT METHODS 2019; 15:105. [PMID: 31516543 PMCID: PMC6731622 DOI: 10.1186/s13007-019-0487-8] [Citation(s) in RCA: 71] [Impact Index Per Article: 11.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/22/2019] [Accepted: 08/20/2019] [Indexed: 05/15/2023]
Abstract
Type 2 Diabetes Mellitus has reached epidemic proportions as a result of over-nutrition and increasingly sedentary lifestyles. Current therapies, although effective, are not without limitations. These limitations, the alarming increase in the prevalence of diabetes, and the soaring cost of managing diabetes and its complications underscores an urgent need for safer, more efficient and affordable alternative treatments. Over 1200 plant species are reported in ethnomedicine for treating diabetes and these represents an important and promising source for the identification of novel antidiabetic compounds. Evaluating medicinal plants for desirable bioactivity goes hand-in-hand with methods in analytical biochemistry for separating and identifying lead compounds. This review aims to provide a comprehensive summary of current methods used in antidiabetic plant research to form a useful resource for researchers beginning in the field. The review summarises the current understanding of blood glucose regulation and the general mechanisms of action of current antidiabetic medications, and combines knowledge on common experimental approaches for screening plant extracts for antidiabetic activity and currently available analytical methods and technologies for the separation and identification of bioactive natural products. Common in vivo animal models, in vitro models, in silico methods and biochemical assays used for testing the antidiabetic effects of plants are discussed with a particular emphasis on in vitro methods such as cell-based bioassays for screening insulin secretagogues and insulinomimetics. Enzyme inhibition assays and molecular docking are also highlighted. The role of metabolomics, metabolite profiling, and dereplication of data for the high-throughput discovery of novel antidiabetic agents is reviewed. Finally, this review also summarises sample preparation techniques such as liquid-liquid extraction, solid phase extraction, and supercritical fluid extraction, and the critical function of nuclear magnetic resonance and high resolution liquid chromatography-mass spectrometry for the dereplication, putative identification and structure elucidation of natural compounds from evidence-based medicinal plants.
Collapse
Affiliation(s)
- Chintha Lankatillake
- School of Health and Biomedical Sciences, Discipline of Laboratory Medicine, RMIT University, Bundoora, 3083 Australia
| | - Tien Huynh
- School of Science, RMIT University, Bundoora, VIC 3083 Australia
| | - Daniel A. Dias
- School of Health and Biomedical Sciences, Discipline of Laboratory Medicine, RMIT University, Bundoora, 3083 Australia
| |
Collapse
|
|
35
|
Torresi J, Tran BM, Christiansen D, Earnest-Silveira L, Schwab RHM, Vincan E. HBV-related hepatocarcinogenesis: the role of signalling pathways and innovative ex vivo research models. BMC Cancer 2019; 19:707. [PMID: 31319796 PMCID: PMC6637598 DOI: 10.1186/s12885-019-5916-6] [Citation(s) in RCA: 66] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2019] [Accepted: 07/09/2019] [Indexed: 12/22/2022] Open
Abstract
BACKGROUND Hepatitis B virus (HBV) is the leading cause of liver cancer, but the mechanisms by which HBV causes liver cancer are poorly understood and chemotherapeutic strategies to cure liver cancer are not available. A better understanding of how HBV requisitions cellular components in the liver will identify novel therapeutic targets for HBV associated hepatocellular carcinoma (HCC). MAIN BODY The development of HCC involves deregulation in several cellular signalling pathways including Wnt/FZD/β-catenin, PI3K/Akt/mTOR, IRS1/IGF, and Ras/Raf/MAPK. HBV is known to dysregulate several hepatocyte pathways and cell cycle regulation resulting in HCC development. A number of these HBV induced changes are also mediated through the Wnt/FZD/β-catenin pathway. The lack of a suitable human liver model for the study of HBV has hampered research into understanding pathogenesis of HBV. Primary human hepatocytes provide one option; however, these cells are prone to losing their hepatic functionality and their ability to support HBV replication. Another approach involves induced-pluripotent stem (iPS) cell-derived hepatocytes. However, iPS technology relies on retroviruses or lentiviruses for effective gene delivery and pose the risk of activating a range of oncogenes. Liver organoids developed from patient-derived liver tissues provide a significant advance in HCC research. Liver organoids retain the characteristics of their original tissue, undergo unlimited expansion, can be differentiated into mature hepatocytes and are susceptible to natural infection with HBV. CONCLUSION By utilizing new ex vivo techniques like liver organoids it will become possible to develop improved and personalized therapeutic approaches that will improve HCC outcomes and potentially lead to a cure for HBV.
Collapse
Affiliation(s)
- Joseph Torresi
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010 Australia
| | - Bang Manh Tran
- The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010 Australia
| | - Dale Christiansen
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010 Australia
| | - Linda Earnest-Silveira
- Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010 Australia
| | - Renate Hilda Marianne Schwab
- The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010 Australia
| | - Elizabeth Vincan
- The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010 Australia
- Victorian Infectious Diseases Reference Laboratory, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Parkville, Victoria 3010 Australia
- School of Pharmacy and Biomedical Sciences, Curtin University, Perth, WA 6845 Australia
| |
Collapse
|
|
36
|
Nagarajan SR, Paul-Heng M, Krycer JR, Fazakerley DJ, Sharland AF, Hoy AJ. Lipid and glucose metabolism in hepatocyte cell lines and primary mouse hepatocytes: a comprehensive resource for in vitro studies of hepatic metabolism. Am J Physiol Endocrinol Metab 2019; 316:E578-E589. [PMID: 30694691 DOI: 10.1152/ajpendo.00365.2018] [Citation(s) in RCA: 69] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/09/2023]
Abstract
The liver is a critical tissue for maintaining glucose, fatty acid, and cholesterol homeostasis. Primary hepatocytes represent the gold standard for studying the mechanisms controlling hepatic glucose, lipid, and cholesterol metabolism in vitro. However, access to primary hepatocytes can be limiting, and therefore, other immortalized hepatocyte models are commonly used. Here, we describe substrate metabolism of cultured AML12, IHH, and PH5CH8 cells, hepatocellular carcinoma-derived HepG2s, and primary mouse hepatocytes (PMH) to identify which of these cell lines most accurately phenocopy PMH basal and insulin-stimulated metabolism. Insulin-stimulated glucose metabolism in PH5CH8 cells, and to a lesser extent AML12 cells, responded most similarly to PMH. Notably, glucose incorporation in HepG2 cells were 14-fold greater than PMH. The differences in glucose metabolic activity were not explained by differential protein expression of key regulators of these pathways, for example glycogen synthase and glycogen content. In contrast, fatty acid metabolism in IHH cells was the closest to PMHs, yet insulin-responsive fatty acid metabolism in AML12 and HepG2 cells was most similar to PMH. Finally, incorporation of acetate into intracellular-free cholesterol was comparable for all cells to PMH; however, insulin-stimulated glucose conversion into lipids and the incorporation of acetate into intracellular cholesterol esters were strikingly different between PMHs and all tested cell lines. In general, AML12 cells most closely phenocopied PMH in vitro energy metabolism. However, the cell line most representative of PMHs differed depending on the mode of metabolism being investigated, and so careful consideration is needed in model selection.
Collapse
Affiliation(s)
- Shilpa R Nagarajan
- Discipline of Physiology, School of Medical Sciences & Bosch Institute, Charles Perkins Centre, Faculty of Medicine and Health, The University of Sydney , New South Wales , Australia
| | - Moumita Paul-Heng
- Discipline of Surgery, Central Clinical School & Bosch Institute, Charles Perkins Centre, Faculty of Medicine and Health, University of Sydney , New South Wales , Australia
| | - James R Krycer
- School of Life and Environmental Sciences, Charles Perkins Centre, Faculty of Science, The University of Sydney , New South Wales , Australia
| | - Daniel J Fazakerley
- School of Life and Environmental Sciences, Charles Perkins Centre, Faculty of Science, The University of Sydney , New South Wales , Australia
| | - Alexandra F Sharland
- Discipline of Surgery, Central Clinical School & Bosch Institute, Charles Perkins Centre, Faculty of Medicine and Health, University of Sydney , New South Wales , Australia
| | - Andrew J Hoy
- Discipline of Physiology, School of Medical Sciences & Bosch Institute, Charles Perkins Centre, Faculty of Medicine and Health, The University of Sydney , New South Wales , Australia
| |
Collapse
|
|
37
|
Kyffin JA, Sharma P, Leedale J, Colley HE, Murdoch C, Mistry P, Webb SD. Impact of cell types and culture methods on the functionality of in vitro liver systems - A review of cell systems for hepatotoxicity assessment. Toxicol In Vitro 2018; 48:262-275. [PMID: 29408671 DOI: 10.1016/j.tiv.2018.01.023] [Citation(s) in RCA: 41] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2017] [Revised: 01/26/2018] [Accepted: 01/27/2018] [Indexed: 12/21/2022]
Abstract
Xenobiotic safety assessment is an area that impacts a multitude of different industry sectors such as medicinal drugs, agrochemicals, industrial chemicals, cosmetics and environmental contaminants. As such there are a number of well-developed in vitro, in vivo and in silico approaches to evaluate their properties and potential impact on the environment and to humans. Additionally, there is the continual investment in multidisciplinary scientists to explore non-animal surrogate technologies to predict specific toxicological outcomes and to improve our understanding of the biological processes regarding the toxic potential of xenobiotics. Here we provide a concise, critical evaluation of a number of in vitro systems utilised to assess the hepatotoxic potential of xenobiotics.
Collapse
Affiliation(s)
- Jonathan A Kyffin
- Department of Applied Mathematics, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool L3 3AF, United Kingdom
| | - Parveen Sharma
- MRC Centre for Drug Safety Science, Department of Molecular and Clinical Pharmacology, Sherrington Building, Ashton Street, University of Liverpool, L69 3GE, United Kingdom.
| | - Joseph Leedale
- EPSRC Liverpool Centre for Mathematics in Healthcare, Department of Mathematical Sciences, Peach Street, University of Liverpool, L69 7ZL, United Kingdom
| | - Helen E Colley
- School of Clinical Dentistry, Claremont Crescent, University of Sheffield, Sheffield S10 2TA, United Kingdom
| | - Craig Murdoch
- School of Clinical Dentistry, Claremont Crescent, University of Sheffield, Sheffield S10 2TA, United Kingdom
| | - Pratibha Mistry
- Syngenta Ltd., Jealott's Hill International Research Centre, Bracknell, Berkshire RG42 6EY, United Kingdom
| | - Steven D Webb
- Department of Applied Mathematics, Liverpool John Moores University, James Parsons Building, Byrom Street, Liverpool L3 3AF, United Kingdom
| |
Collapse
|
|
38
|
Hang HL, Liu XY, Wang HT, Xu N, Bian JM, Zhang JJ, Xia L, Xia Q. Hepatocyte nuclear factor 4A improves hepatic differentiation of immortalized adult human hepatocytes and improves liver function and survival. Exp Cell Res 2017; 360:81-93. [PMID: 28870599 DOI: 10.1016/j.yexcr.2017.08.020] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2017] [Revised: 08/10/2017] [Accepted: 08/12/2017] [Indexed: 12/22/2022]
Abstract
Immortalized human hepatocytes (IHH) could provide an unlimited supply of hepatocytes, but insufficient differentiation and phenotypic instability restrict their clinical application. This study aimed to determine the role of hepatocyte nuclear factor 4A (HNF4A) in hepatic differentiation of IHH, and whether encapsulation of IHH overexpressing HNF4A could improve liver function and survival in rats with acute liver failure (ALF). Primary human hepatocytes were transduced with lentivirus-mediated catalytic subunit of human telomerase reverse transcriptase (hTERT) to establish IHH. Cells were analyzed for telomerase activity, proliferative capacity, hepatocyte markers, and tumorigenicity (c-myc) expression. Hepatocyte markers, hepatocellular functions, and morphology were studied in the HNF4A-overexpressing IHH. Hepatocyte markers and karyotype analysis were completed in the primary hepatocytes using shRNA knockdown of HNF4A. Nuclear translocation of β-catenin was assessed. Rat models of ALF were treated with encapsulated IHH or HNF4A-overexpressing IHH. A HNF4A-positive IHH line was established, which was non-tumorigenic and conserved properties of primary hepatocytes. HNF4A overexpression significantly enhanced mRNA levels of genes related to hepatic differentiation in IHH. Urea levels were increased by the overexpression of HNF4A, as measured 24h after ammonium chloride addition, similar to that of primary hepatocytes. Chromosomal abnormalities were observed in primary hepatocytes transfected with HNF4A shRNA. HNF4α overexpression could significantly promote β-catenin activation. Transplantation of HNF4A overexpressing IHH resulted in better liver function and survival of rats with ALF compared with IHH. HNF4A improved hepatic differentiation of IHH. Transplantation of HNF4A-overexpressing IHH could improve the liver function and survival in a rat model of ALF.
Collapse
Affiliation(s)
- Hua-Lian Hang
- Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
| | - Xin-Yu Liu
- Department of General Surgery, Nanjing Hospital Affiliated to NanJing Medical University, Nanjing 210006, China
| | - Hai-Tian Wang
- Department of General Surgery, Nanjing Hospital Affiliated to NanJing Medical University, Nanjing 210006, China
| | - Ning Xu
- Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
| | - Jian-Min Bian
- Department of General Surgery, Nanjing Hospital Affiliated to NanJing Medical University, Nanjing 210006, China
| | - Jian-Jun Zhang
- Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China
| | - Lei Xia
- Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.
| | - Qiang Xia
- Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200127, China.
| |
Collapse
|
|
39
|
Tubulin alpha 8 is expressed in hepatic stellate cells and is induced in transformed hepatocytes. Mol Cell Biochem 2017; 428:161-170. [DOI: 10.1007/s11010-016-2926-4] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2016] [Accepted: 12/21/2016] [Indexed: 12/16/2022]
|
|
40
|
Wiśniewski JR, Vildhede A, Norén A, Artursson P. In-depth quantitative analysis and comparison of the human hepatocyte and hepatoma cell line HepG2 proteomes. J Proteomics 2016; 136:234-47. [PMID: 26825538 DOI: 10.1016/j.jprot.2016.01.016] [Citation(s) in RCA: 133] [Impact Index Per Article: 14.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/09/2015] [Revised: 11/28/2015] [Accepted: 01/25/2016] [Indexed: 12/14/2022]
Abstract
Hepatocytes play a pivotal role in human homeostasis. They are essential in regulation of glucose and lipid levels in blood and play a central role in metabolism of amino acids, lipids, drugs and xenobiotic-compounds. In addition, hepatocytes produce a major portion of proteins circulating in the blood. Hepatocytes were isolated from liver tissue obtained from surgical resections. Proteins were extracted and processed using filter aided sample preparation protocol and were analyzed by LC-MS/MS using high accuracy mass spectrometry. Proteins were quantified by the 'Total Protein Approach' and 'Proteomic Ruler'. We report a comprehensive proteomic analysis of purified human hepatocytes and the human hepatoma cell line HepG2. The complete dataset comprises 9400 proteins and provides a comprehensive and quantitative depiction of the proteomes of hepatocytes and HepG2 cells at the protein titer and copy number dimensions. We describe basic cell organization and in detail energy metabolism pathways and metabolite transport. We provide quantitative insights into protein synthesis and drug and xenobiotics catabolism. Our data delineate differences between the native human hepatocytes and HepG2 cells by providing for the first time quantitative data at protein concentrations and copy numbers.
Collapse
Affiliation(s)
- Jacek R Wiśniewski
- Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
| | - Anna Vildhede
- Department of Pharmacy, Uppsala University, Uppsala, Sweden
| | - Agneta Norén
- Department of Surgery, Uppsala University, Uppsala, Sweden
| | - Per Artursson
- Department of Pharmacy, Uppsala University, Uppsala, Sweden; Science for Life Laboratory, Uppsala, Sweden
| |
Collapse
|