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

Marc Ruoß,Georg Damm,Andreas Nussler,Carl Grom-Baumgarten,Lisa Ehret,Ruth Ladurner,Silvio Naddalin,Daniel Seehofer,Sahar Sajadian,Martin Petkov,Massoud Vosough

doi:10.3390/ijms20020347

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.

Highlights

Drug metabolism is understood to mean the biochemical process which describes the modification of drugs, which has the purpose to inactivate a substance and excrete it from the body
Our results clearly show that HepG2 treatment with 5-AZA plus Vitamin C led to a significant reduction of the Epithelial-mesenchymal transition (EMT) marker gene SNAIL and a significant increase of E-Cadherin gene expression, which is in line with other human cancer cell lines [29]
Our results show that the epigenetic status of the hepatoblastoma cell line HepG2 shows the highest similarity with primary human hepatocytes (PHH) compared to the other tested liver cancer cell lines (Huh7, HLE and AKN1)

Summary

Introduction

Drug metabolism is understood to mean the biochemical process which describes the modification of drugs, which has the purpose to inactivate a substance and excrete it from the body. Changes in the gene expression of enzymes specialized in drug metabolism may result in altered metabolism of the respective substance [1] In recent years, it has been shown in many studies that the epigenetic regulation of drug-metabolizing enzymes is an important mechanism here. Recent studies have revealed that epigenetic factors regulate the expression of drug-metabolizing enzymes and the drug transporter [1,3]. It was shown that the most important hepatic genes like Hepatocyte nuclear factor 4α (HNF4α) are influenced by epigenetic regulators such as HDACi (Histone deacetylase inhibitors) and DNMTi (DNA methyltransferase inhibitors) [10]. Epigenetic modification promotes growth arrest and up-regulates the expression of the hepatic key regulator gene HNF4α in various hepatoma cells which induces increased CYP expression and Albumin production [11]. It is known that insulin contributes to the preservation of hepatocytes morphology and the glucocorticoids support the maintenance of differentiation which is crucial for the function of CYPs [13,14]

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