Azazytidin and Vitamin C modulates the expression profile of epigenetic modulators in HepG2 and increase their metabolic function

Background:

Drug-Induced Hepatotoxicity is the leading cause of acute liver failure and Post-marketing withdrawal of drugs. Preclinical animal studies cannot fully predict drug-toxicity in humans due to species-specific variations in hepatocellular functions between human and animals. Today human hepatocytes are the gold standard for metabolism and toxicity testing of new pharmaceuticals. However, PHHs have limitations, such as scarce availability and rapid de-differentiation in culture. Therefore, we used HEPG2-cells as an alternative, though its drug metabolizing activity is low. Until today, many attempts at improving the drug metabolizing activity of HEPG2 cells have failed. Studies have shown that epigenetic regulators influence the expression and function of some of the phase I/II enzymes by altering crucial transcription factors. Thus, we attempted to modify the epigenetic status of these cells by 5-Azacytidine (5-AZA) and Vitamin C treatment in order to improve their metabolic status.

Methods:

HEPG2 cells were stimulated with 5-AZA and Vitamin C containing media, the expression of epigenetic modifying enzymes were quantified by using a Human Epigenetic Chromatin Modification Enzymes PCR Array. Additionally RNA/Protein expression of EMT markers and Phase I/II enzymes were quantified using qPCR/WB. Subsequently, the activity of some Phase I/II enzymes were tested.

Results:

The treatment of HEPG2-cells with 5-AZA and Vitamin C results in a global change in chromatin modifying enzymes, these epigenetic modifications result in a changed expression of HNF4α, CK18 and E-Cadherin, which are key regulators of liver-specific gene expression. Similarly, the EMT marker-gene Snail significantly decreased after stimulation. Furthermore, the mRNA expression and the metabolic activity of some Phase I/II enzymes are significantly increased after stimulation with 5-AZA and/or Vitamin C.

Conclusions:

Epigenetic modification of HEPG2-cells resulted in increased Phase I/II gene expression and enzyme activity. This increase was accompanied by a global change in the expression of some epigenetic enzymes and a reduction of EMT. Enhancement of liver specific functions in partially metabolic expressing hepatocellular tumour cells using epigenetic modifiers may open new opportunities for toxicity screening in drug development.