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

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.

[1]  F. Schügner,et al.  A Standardized Collagen-Based Scaffold Improves Human Hepatocyte Shipment and Allows Metabolic Studies over 10 Days , 2018, Bioengineering.

[2]  M. Bakhtiyari,et al.  The effects of hydrocortisone on tight junction genes in an in vitro model of the human fallopian epithelial cells. , 2018, European journal of obstetrics, gynecology, and reproductive biology.

[3]  Miao Yin,et al.  CARM1 Methylates GAPDH to Regulate Glucose Metabolism and Is Suppressed in Liver Cancer. , 2018, Cell reports.

[4]  C. Frezza,et al.  Metabolic reprogramming and epithelial‐to‐mesenchymal transition in cancer , 2017, The FEBS journal.

[5]  F. Filipp.  Crosstalk between epigenetics and metabolism—Yin and Yang of histone demethylases and methyltransferases in cancer , 2017, Briefings in functional genomics.

[6]  A. Mukhopadhyay,et al.  JMJD3 aids in reprogramming of bone marrow progenitor cells to hepatic phenotype through epigenetic activation of hepatic transcription factors , 2017, PloS one.

[7]  Q. Gou,et al.  Protein arginine N-methyltransferase 1 promotes the proliferation and metastasis of hepatocellular carcinoma cells , 2017, Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine.

[8]  S. Dooley,et al.  Vitamin C enhances epigenetic modifications induced by 5-azacytidine and cell cycle arrest in the hepatocellular carcinoma cell lines HLE and Huh7 , 2016, Clinical Epigenetics.

[9]  S. Alahari,et al.  Regulation of epithelial-mesenchymal transition through epigenetic and post-translational modifications , 2016, Molecular Cancer.

[10]  S. Dooley,et al.  Induction of active demethylation and 5hmC formation by 5-azacytidine is TET2 dependent and suggests new treatment strategies against hepatocellular carcinoma , 2015, Clinical Epigenetics.

[11]  Seok-joo Yoon,et al.  Differences in the Epigenetic Regulation of Cytochrome P450 Genes between Human Embryonic Stem Cell-Derived Hepatocytes and Primary Hepatocytes , 2015, PloS one.

[12]  J. Hengstler,et al.  Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells , 2015, Experimental biology and medicine.

[13]  Xiaobo Zhong,et al.  Epigenetic regulation of drug metabolism and transport , 2015, Acta pharmaceutica Sinica. B.

[14]  J. Hengstler,et al.  Lineage-Specific Regulation of Epigenetic Modifier Genes in Human Liver and Brain , 2014, PloS one.

[15]  Min Liu,et al.  DNA Methylation-mediated Repression of miR-941 Enhances Lysine (K)-specific Demethylase 6B Expression in Hepatoma Cells* , 2014, The Journal of Biological Chemistry.

[16]  Hong Lu,et al.  Alterations of Epigenetic Signatures in Hepatocyte Nuclear Factor 4α Deficient Mouse Liver Determined by Improved ChIP-qPCR and (h)MeDIP-qPCR Assays , 2014, PloS one.

[17]  M. Ingelman-Sundberg,et al.  Potential Role of Epigenetic Mechanisms in the Regulation of Drug Metabolism and Transport , 2013, Drug Metabolism and Disposition.

[18]  Jun Cheng,et al.  The Up-Regulation of Histone Deacetylase 8 Promotes Proliferation and Inhibits Apoptosis in Hepatocellular Carcinoma , 2013, Digestive Diseases and Sciences.

[19]  J. Hengstler,et al.  Decrease of Global Methylation Improves Significantly Hepatic Differentiation of Ad-MSCs: Possible Future Application for Urea Detoxification , 2013, Cell transplantation.

[20]  U. Müller-Vieira,et al.  Analysis of drug metabolism activities in a miniaturized liver cell bioreactor for use in pharmacological studies , 2012, Biotechnology and bioengineering.

[21]  C. Allis,et al.  Linking epithelial-to-mesenchymal-transition and epigenetic modifications. , 2012, Seminars in cancer biology.

[22]  Jun Yao,et al.  G9a interacts with Snail and is critical for Snail-mediated E-cadherin repression in human breast cancer. , 2012, The Journal of clinical investigation.

[23]  Yun Cui,et al.  Biological Functions of Cytokeratin 18 in Cancer , 2012, Molecular Cancer Research.

[24]  H. Sitter,et al.  Clinical significance of histone deacetylases 1, 2, 3, and 7: HDAC2 is an independent predictor of survival in HCC , 2011, Virchows Archiv.

[25]  R. Huang,et al.  Epithelial-Mesenchymal Transitions in Development and Disease , 2009, Cell.

[26]  S. Cheung,et al.  Hep G2 is a hepatoblastoma-derived cell line. , 2009, Human pathology.

[27]  Vera Rogiers,et al.  Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation. , 2009, Journal of hepatology.

[28]  K. Helin,et al.  The H3K27me3 demethylase JMJD3 contributes to the activation of the INK4A-ARF locus in response to oncogene- and stress-induced senescence. , 2009, Genes & development.

[29]  F. Spagnoli,et al.  Snail controls differentiation of hepatocytes by repressing HNF4α expression , 2006 .

[30]  Luke O Dannenberg,et al.  Epigenetics of gene expression in human hepatoma cells: expression profiling the response to inhibition of DNA methylation and histone deacetylation , 2006, BMC Genomics.

[31]  Lung-Ji Chang,et al.  De novo DNA methyltransferases Dnmt3a and Dnmt3b primarily mediate the cytotoxic effect of 5-aza-2′-deoxycytidine , 2005, Oncogene.

[32]  Peter A. Jones,et al.  Epigenetics in human disease and prospects for epigenetic therapy , 2004, Nature.

[33]  K. Sugimachi,et al.  Transcriptional repressor snail and progression of human hepatocellular carcinoma. , 2003, Clinical cancer research : an official journal of the American Association for Cancer Research.

[34]  K. Sugimachi,et al.  Histone deacetylase inhibitor trichostatin a induces cell‐cycle arrest/apoptosis and hepatocyte differentiation in human hepatoma cells , 2003, International journal of cancer.

[35]  A. Miyajima,et al.  Cytokine regulation of liver development. , 2002, Biochimica et biophysica acta.

[36]  R. Edwards,et al.  Cytochrome P450 expression in human hepatocytes and hepatoma cell lines: molecular mechanisms that determine lower expression in cultured cells , 2002, Xenobiotica; the fate of foreign compounds in biological systems.

[37]  Michael A. Choti,et al.  A Phosphatase Associated with Metastasis of Colorectal Cancer , 2001, Science.

[38]  S. Strom,et al.  Isolation and characterization of a human hepatic epithelial-like cell line (AKN-1) from a normal liver , 1999, In Vitro Cellular & Developmental Biology - Animal.

[39]  D. Scudiero,et al.  New colorimetric cytotoxicity assay for anticancer-drug screening. , 1990, Journal of the National Cancer Institute.

[40]  T. Yamane,et al.  Growth of human hepatoma cells lines with differentiated functions in chemically defined medium. , 1982, Cancer research.

[41]  M. Namba,et al.  Establishment and some biological characteristics of human hepatoma cell lines. , 1975, Gan.

[42]  G. Michalopoulos,et al.  Primary culture of parenchymal liver cells on collagen membranes. Morphological and biochemical observations. , 1975, Experimental cell research.

[43]  J. Hengstler,et al.  Human hepatocytes: isolation, culture, and quality procedures. , 2012, Methods in molecular biology.

[44]  S. Dooley,et al.  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 , 2011, Archives of Toxicology.

[45]  F. Spagnoli,et al.  Snail controls differentiation of hepatocytes by repressing HNF4alpha expression. , 2006, Journal of cellular physiology.