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Cited by 37Pharmacogenetics and Drug MetabolismEpigenetics and DNA MethylationDrug-Induced Hepatotoxicity and Protection

Epigenetic Modifications of the Liver Tumor Cell Line HepG2 Increase Their Drug Metabolic Capacity

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Ruoß, Marc, Damm, Georg, Vosough, Massoud, Ehret, Lisa, Grom-Baumgarten, Carl, Petkov, Martin, Naddalin, Silvio, Ladurner, Ruth +3 more·2019·International Journal of Molecular Sciences
DOI 10.3390/ijms20020347PMC PMC6358789
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Abstract

Although human liver tumor cells have reduced metabolic functions as compared to primary human hepatocytes (PHH) they are widely used for pre-screening tests of drug metabolism and toxicity. The aim of the present study was to modify liver cancer cell lines in order to improve their drug-metabolizing activities towards PHH. It is well-known that epigenetics is strongly modified in tumor cells and that epigenetic regulators influence the expression and function of Cytochrome P450 (CYP) enzymes through altering crucial transcription factors responsible for drug-metabolizing enzymes. Therefore, we screened the epigenetic status of four different liver cancer cell lines (Huh7, HLE, HepG2 and AKN-1) which were reported to have metabolizing drug activities. Our results showed that HepG2 cells demonstrated the highest similarity compared to PHH. Thus, we modified the epigenetic status of HepG2 cells towards ‘normal’ liver cells by 5-Azacytidine (5-AZA) and Vitamin C exposure. Then, mRNA expression of Epithelial-mesenchymal transition (EMT) marker SNAIL and CYP enzymes were measured by PCR and determinate specific drug metabolites, associated with CYP enzymes by LC/MS. Our results demonstrated an epigenetic shift in HepG2 cells towards PHH after exposure to 5-AZA and Vitamin C which resulted in a higher expression and activity of specific drug metabolizing CYP enzymes. Finally, we observed that 5-AZA and Vitamin C led to an increased expression of Hepatocyte nuclear factor 4α (HNF4α) and E-Cadherin and a significant down regulation of Snail1 (SNAIL), the key transcriptional repressor of E-Cadherin. Our study shows, that certain phase I genes and their enzyme activities are increased by epigenetic modification in HepG2 cells with a concomitant reduction of EMT marker gene SNAIL. The enhancing of liver specific functions in hepatoma cells using epigenetic modifiers opens new opportunities for the usage of cell lines as a potential liver in vitro model for drug testing and development.

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  • Epigenetic reprogramming using 5-azacytidine, vitamin C, and hormonal supplements (insulin and hydrocortisone) partially shifts HepG2 cells toward a more differentiated hepatic phenotype and increases the expression of certain CYP450 enzymes (CYP1A2, CYP2C9, CYP3A4), but enzymatic activity increases only slightly (statistically insignificant) and, as a rule, does not reach PHH levels.

    “Epigenetic reprogramming using 5-azacytidine, vitamin C, and hormonal supplements (insulin and hydrocortisone) partially shifts HepG2 cells toward a more differentiated hepatic phenotype and increases the expression of certain CYP450 enzymes (CYP1A2, CYP2C9, CYP3A4), but enzymatic activity increases only slightly (statistically insignificant) and, as a rule, does not reach PHH levels [162].”

    From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [162]

  • Strategies for optimizing HepG2 can be divided into genetic modification to express key biotransformation enzymes, epigenetic and cultural maturation, external metabolic activation using subcellular liver fractions (S9 or microsomes), and transitioning from monolayer culture to 3D or hybrid formats.

    “In practice, strategies for optimizing HepG2 can be divided into several approaches. The first approach is based on genetic modification of cells to express key biotransformation enzymes, in particular cytochrome P450 isoforms [109]. The second approach involves epigenetic and cultural maturation of cells, which allows for increased expression of nuclear receptors and, consequently, of xenobiotic”

    From The HepG2 Cell Line as a Model for Studying Metabolic Dysfunction-Associated Steatotic Liver Disease (2026) · cites this paper as [162]

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References 46 · Cited by 37

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  1. [1]Peng L. Zhong X. Epigenetic regulation of drug metabolism and transport Acta Pharm. Sin. B 2015 5 106 112 10.1016/j.apsb.2015.01.007 26579435 PMC4629221 · DOI
  2. [2]Egger G. Liang G. Aparicio A. Jones P.A. Epigenetics in human disease and prospects for epigenetic therapy Nature 2004 429 457 463 10.1038/nature02625 15164071 · DOI · Paper page
  3. [3]Ingelman-Sundberg M. Zhong X.B. Hankinson O. Beedanagari S. Yu A.M. Peng L. Osawa Y. Potential role of epigenetic mechanisms in the regulation of drug metabolism and transport Drug. Metab. Dispos. 2013 41 1725 1731 10.1124/dmd.113.053157 23918665 PMC3781370 · DOI
  4. [4]Park H.J. Choi Y.J. Kim J.W. Chun H.S. Im I. Yoon S. Han Y.M. Song C.W. Kim H. Differences in the epigenetic regulation of cytochrome p450 genes between human embryonic stem cell-derived hepatocytes and primary hepatocytes PLoS ONE 2015 10 e0132992 10.1371/journal.pone.0132992 26177506 PMC4503736 · DOI
  5. [5]Stadler S.C. Allis C.D. Linking epithelial-to-mesenchymal-transition and epigenetic modifications Semin. Cancer Biol. 2012 22 404 410 10.1016/j.semcancer.2012.06.007 22706095 PMC3445725 · DOI
  6. [6]Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease Cell 2009 139 871 890 10.1016/j.cell.2009.11.007 19945376 · DOI · Paper page
  7. [7]Sciacovelli M. Frezza C. Metabolic reprogramming and epithelial-to-mesenchymal transition in cancer FEBS J. 2017 284 3132 3144 10.1111/febs.14090 28444969 PMC6049610 · DOI
  8. [8]Serrano-Gomez S.J. Maziveyi M. Alahari S.K. Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications Mol. Cancer 2016 15 18 10.1186/s12943-016-0502-x 26905733 PMC4765192 · DOI
  9. [9]Cicchini C. Filippini D. Coen S. Marchetti A. Cavallari C. Laudadio I. Spagnoli F.M. Alonzi T. Tripodi M. Snail controls differentiation of hepatocytes by repressing hnf4alpha expression J. Cell Physiol. 2006 209 230 238 10.1002/jcp.20730 16826572 · DOI
  10. [10]Snykers S. Henkens T. De Rop E. Vinken M. Fraczek J. De Kock J. De Prins E. Geerts A. Rogiers V. Vanhaecke T. Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation J. Hepatol. 2009 51 187 211 10.1016/j.jhep.2009.03.009 19457566 · DOI
  11. [11]Yamashita Y. Shimada M. Harimoto N. Rikimaru T. Shirabe K. Tanaka S. Sugimachi K. Histone deacetylase inhibitor trichostatin a induces cell-cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells Int. J. Cancer 2003 103 572 576 10.1002/ijc.10699 12494463 · DOI
  12. [12]Sajadian S.O. Tripura C. Samani F.S. Ruoss M. Dooley S. Baharvand H. Nussler A.K. Vitamin c enhances epigenetic modifications induced by 5-azacytidine and cell cycle arrest in the hepatocellular carcinoma cell lines hle and huh7 Clin. Epigenetics. 2016 8 46 10.1186/s13148-016-0213-6 27134688 PMC4851801 · DOI
  13. [13]Michalopoulos G. Pitot H.C. Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations Exp. Cell Res. 1975 94 70 78 10.1016/0014-4827(75)90532-7 243 · DOI
  14. [14]Kinoshita T. Miyajima A. Cytokine regulation of liver development Biochim. Biophys. Acta 2002 1592 303 312 10.1016/S0167-4889(02)00323-3 12421674 · DOI
  15. [15]Lin J. Schyschka L. Muhl-Benninghaus R. Neumann J. Hao L. Nussler N. Dooley S. Liu L. Stockle U. Nussler A.K. Comparative analysis of phase i and ii enzyme activities in 5 hepatic cell lines identifies huh-7 and hcc-t cells with the highest potential to study drug metabolism Arch. Toxicol. 2012 86 87 95 10.1007/s00204-011-0733-y 21735230 · DOI
  16. [16]Nussler A.K. Vergani G. Gollin S.M. Dorko K. Morris S.M. Jr. Demetris A.J. Nomoto M. Beger H.G. Strom S.C. Isolation and characterization of a human hepatic epithelial-like cell line (akn-1) from a normal liver In. Vitro Cell Dev. Biol. Anim. 1999 35 190 197 10.1007/s11626-999-0026-4 10478798 · DOI
  17. [17]Ruoß M. Häussling V. Schügner F. Olde Damink L. Lee S. Ge L. Ehnert S. Nussler A. A standardized collagen-based scaffold improves human hepatocyte shipment and allows metabolic studies over 10 days Bioengineering 2018 5 86 10.3390/bioengineering5040086 PMC6316810 30332824 · DOI
  18. [18]Rodriguez-Antona C. Donato M.T. Boobis A. Edwards R.J. Watts P.S. Castell J.V. Gomez-Lechon M.J. Cytochrome p450 expression in human hepatocytes and hepatoma cell lines: Molecular mechanisms that determine lower expression in cultured cells Xenobiotica 2002 32 505 520 10.1080/00498250210128675 12160483 · DOI · Paper page
  19. [19]Seeliger C. Culmes M. Schyschka L. Yan X. Damm G. Wang Z. Kleeff J. Thasler W.E. Hengstler J. Stockle U. Decrease of global methylation improves significantly hepatic differentiation of ad-mscs: Possible future application for urea detoxification Cell Transplant. 2013 22 119 131 10.3727/096368912X638946 22507189 · DOI
  20. [20]Quint K. Agaimy A. Di Fazio P. Montalbano R. Steindorf C. Jung R. Hellerbrand C. Hartmann A. Sitter H. Neureiter D. Clinical significance of histone deacetylases 1, 2, 3, and 7: Hdac2 is an independent predictor of survival in hcc Virchows Arch. 2011 459 129 139 10.1007/s00428-011-1103-0 21713366 · DOI
  21. [21]Gou Q. He S. Zhou Z. Protein arginine n-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells Tumour Biol. 2017 39 1010428317691419 10.1177/1010428317691419 28231732 · DOI
  22. [22]Filipp F.V. Crosstalk between epigenetics and metabolism-yin and yang of histone demethylases and methyltransferases in cancer Brief Funct. Genomics 2017 16 320 325 10.1093/bfgp/elx001 28369194 PMC5860014 · DOI
  23. [23]Dong C. Wu Y. Yao J. Wang Y. Yu Y. Rychahou P.G. Evers B.M. Zhou B.P. G9a interacts with snail and is critical for snail-mediated e-cadherin repression in human breast cancer J. Clin. Invest. 2012 122 1469 1486 10.1172/JCI57349 22406531 PMC3314447 · DOI
  24. [24]Agger K. Cloos P.A. Rudkjaer L. Williams K. Andersen G. Christensen J. Helin K. The h3k27me3 demethylase jmjd3 contributes to the activation of the ink4a-arf locus in response to oncogene- and stress-induced senescence Genes Dev. 2009 23 1171 1176 10.1101/gad.510809 19451217 PMC2685535 · DOI · Paper page
  25. [25]Zhang P.P. Wang X.L. Zhao W. Qi B. Yang Q. Wan H.Y. Shuang Z.Y. Liu M. Li X. Li S. DNA methylation-mediated repression of mir-941 enhances lysine (k)-specific demethylase 6b expression in hepatoma cells J. Biol. Chem. 2014 289 24724 24735 10.1074/jbc.M114.567818 25049231 PMC4148894 · DOI
  26. [26]Kochat V. Equbal Z. Baligar P. Kumar V. Srivastava M. Mukhopadhyay A. Jmjd3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors PLoS ONE 2017 12 e0173977 10.1371/journal.pone.0173977 28328977 PMC5362104 · DOI
  27. [27]Wu J. Du C. Lv Z. Ding C. Cheng J. Xie H. Zhou L. Zheng S. The up-regulation of histone deacetylase 8 promotes proliferation and inhibits apoptosis in hepatocellular carcinoma Dig. Dis. Sci. 2013 58 3545 3553 10.1007/s10620-013-2867-7 24077923 · DOI
  28. [28]Zhong X.Y. Yuan X.M. Xu Y.Y. Yin M. Yan W.W. Zou S.W. Wei L.M. Lu H.J. Wang Y.P. Lei Q.Y. Carm1 methylates gapdh to regulate glucose metabolism and is suppressed in liver cancer Cell Rep. 2018 24 3207 3223 10.1016/j.celrep.2018.08.066 30232003 · DOI
  29. [29]Sugimachi K. Tanaka S. Kameyama T. Taguchi K. Aishima S. Shimada M. Sugimachi K. Tsuneyoshi M. Transcriptional repressor snail and progression of human hepatocellular carcinoma Clin. Cancer Res. 2003 9 2657 2664 12855644
  30. [30]Zhaeentan S. Amjadi F.S. Zandie Z. Joghataei M.T. Bakhtiyari M. Aflatoonian R. The effects of hydrocortisone on tight junction genes in an in vitro model of the human fallopian epithelial cells Eur. J. Obstet. Gynecol. Reprod. Biol. 2018 229 127 131 10.1016/j.ejogrb.2018.05.034 30173088 · DOI
  31. [31]Zhang Q. Lei X. Lu H. Alterations of epigenetic signatures in hepatocyte nuclear factor 4alpha deficient mouse liver determined by improved chip-qpcr and (h)medip-qpcr assays PLoS ONE 2014 9 e84925 24427299 PMC3888413
  32. [32]Weng Y.R. Cui Y. Fang J.Y. Biological functions of cytokeratin 18 in cancer Mol. Cancer Res. 2012 10 485 493 10.1158/1541-7786.MCR-11-0222 22452884 · DOI
  33. [33]Dannenberg L.O. Edenberg H.J. Epigenetics of gene expression in human hepatoma cells: Expression profiling the response to inhibition of DNA methylation and histone deacetylation BMC Genomics 2006 7 181 10.1186/1471-2164-7-181 16854234 PMC1574318 · DOI
  34. [34]Weng M.K. Natarajan K. Scholz D. Ivanova V.N. Sachinidis A. Hengstler J.G. Waldmann T. Leist M. Lineage-specific regulation of epigenetic modifier genes in human liver and brain PLoS ONE 2014 9 e102035 10.1371/journal.pone.0102035 25054330 PMC4108363 · DOI
  35. [35]Oka M. Meacham A.M. Hamazaki T. Rodic N. Chang L.J. Terada N. De novo DNA methyltransferases dnmt3a and dnmt3b primarily mediate the cytotoxic effect of 5-aza-2′-deoxycytidine Oncogene 2005 24 3091 3099 10.1038/sj.onc.1208540 15735669 · DOI
  36. [36]Pfeiffer E. Kegel V. Zeilinger K. Hengstler J.G. Nussler A.K. Seehofer D. Damm G. Featured article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells Exp. Biol. Med. 2015 240 645 656 10.1177/1535370214558025 PMC4935273 25394621 · DOI
  37. [37]Knobeloch D. Ehnert S. Schyschka L. Buchler P. Schoenberg M. Kleeff J. Thasler W.E. Nussler N.C. Godoy P. Hengstler J. Human hepatocytes: Isolation, culture, and quality procedures Methods Mol. Biol. 2012 806 99 120 22057448
  38. [38]Sajadian S.O. Ehnert S. Vakilian H. Koutsouraki E. Damm G. Seehofer D. Thasler W. Dooley S. Baharvand H. Sipos B. Induction of active demethylation and 5hmc formation by 5-azacytidine is tet2 dependent and suggests new treatment strategies against hepatocellular carcinoma Clin. Epigenetics. 2015 7 98 10.1186/s13148-015-0133-x 26366235 PMC4567821 · DOI
  39. [39]Lopez-Terrada D. Cheung S.W. Finegold M.J. Knowles B.B. Hep g2 is a hepatoblastoma-derived cell line Hum. Pathol. 2009 40 1512 1515 10.1016/j.humpath.2009.07.003 19751877 · DOI
  40. [40]Nakabayashi H. Taketa K. Miyano K. Yamane T. Sato J. Growth of human hepatoma cells lines with differentiated functions in chemically defined medium Cancer Res. 1982 42 3858 3863 6286115
  41. [41]Doi I. Namba M. Sato J. Establishment and some biological characteristics of human hepatoma cell lines Gann 1975 66 385 392 52570
  42. [42]Saha S. Bardelli A. Buckhaults P. Velculescu V.E. Rago C. St Croix B. Romans K.E. Choti M.A. Lengauer C. Kinzler K.W. A phosphatase associated with metastasis of colorectal cancer Science 2001 294 1343 1346 10.1126/science.1065817 11598267 · DOI
  43. [43]Hoffmann S.A. Muller-Vieira U. Biemel K. Knobeloch D. Heydel S. Lubberstedt M. Nussler A.K. Andersson T.B. Gerlach J.C. Zeilinger K. Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies Biotechnol. Bioeng. 2012 109 3172 3181 10.1002/bit.24573 22688505 · DOI
  44. [44]Skehan P. Storeng R. Scudiero D. Monks A. McMahon J. Vistica D. Warren J.T. Bokesch H. Kenney S. Boyd M.R. New colorimetric cytotoxicity assay for anticancer-drug screening J. Natl. Cancer Inst. 1990 82 1107 1112 10.1093/jnci/82.13.1107 2359136 · DOI

Authors
Ruoß, M., Damm, G., Vosough, M., Ehret, L., Grom-Baumgarten, C., Petkov, M., Naddalin, S., Ladurner, R., Seehofer, D., Nussler, A., & Sajadian, S.
Affiliations
  • University of Tübingen (DE)
  • Leipzig University (DE)
  • Royan Institute (IR)
  • Universitätsklinikum Tübingen (DE)
Corresponding author
Nussler, Andreas
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2019-01-16
Volume
20
Issue
2
Pages
347
ISSN
1422-0067 (ISSN-L)
DOI
10.3390/ijms20020347
PMC
PMC6358789
OpenAlex
W2909104459
Cited by
37
References
46
Citations by year
2026: 6 · 2025: 2 · 2024: 6 · 2023: 4 · 2022: 5 · 2021: 6 · 2020: 6 · 2019: 2
FWCI
4.78
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Molecular Sciences (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Qucosa (Saxon State and University Library Dresden) (submittedVersion, cc-by)
Funders
  • Ministerium für Ländlichen Raum und Verbraucherschutz/Baden-Württemberg (BW 05110214)
  • Deutsche Forschungsgemeinschaft and Open Access Publishing Fund of University of Tübingen (-)
Topics
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Pharmacogenetics and Drug Metabolism
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
  • Life Sciences › Pharmacology, Toxicology and Pharmaceutics › Pharmacology › Drug-Induced Hepatotoxicity and Protection
Keywords
epigenetic modifications, HepG2 cells, liver cancer cell lines, drug metabolism, cytochrome P450, azacitidine, vitamin C, epithelial-mesenchymal transition, snails, HNF4α, E-cadherin, hepatocyte-like cells, in vitro liver models, drug toxicity testing
MeSH terms
Snail Family Transcription Factors, Ascorbic Acid, Azacitidine, Chromatin, Cytochrome P-450 Enzyme System, Pharmaceutical Preparations, Humans, Hydrocortisone, Insulin, Liver Neoplasms, Biomarkers, Tumor, Xenobiotics, Down-Regulation, Gene Expression Regulation, Neoplastic, Hepatocytes, Epigenesis, Genetic, Hepatocyte Nuclear Factor 4, Hep G2 Cells, Epithelial-Mesenchymal Transition

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