Vitamin C enhances epigenetic modifications induced by 5-azacytidine and cell cycle arrest in the hepatocellular carcinoma cell lines HLE and Huh7

Sahar Olsadat Sajadian,Hossein Baharvand,Steven Dooley,Marc Ruoss,Chaturvedula Tripura,Fazel Sahraneshin Samani,Andreas K Nussler

doi:10.1186/s13148-016-0213-6

Abstract

Background5-Azacytidine (5-AZA), a DNA methyl transferase inhibitor, is a clinically used epigenetic drug for cancer therapy. Recently, we have shown that 5-AZA upregulates ten-eleven translocation (TET) protein expression in hepatocellular carcinoma (HCC) cells, which induce active demethylation. Vitamin C facilitates TET activity and enhances active demethylation. The aim of this study is to investigate whether vitamin C is able to enhance the effect of 5-AZA on active demethylation and to evaluate its consequence in HCC cell lines.MethodsHCC cell lines (Huh7 and HLE) were treated with 5-AZA and vitamin C. After 48 h of treatment, viability (resazurin conversion), toxicity (lactose dehydrogenase (LDH) release), and proliferation ((proliferating cell nuclear antigen (PCNA)) of single- and combined-treated cells were assessed. The effect of the treatment on 5-hydroxymethylcytosine (5hmC) intensity (immunofluorescence (IF) staining), TET, Snail, GADD45B, and P21 mRNA (real-time PCR) and protein expression (Western blot) were investigated.ResultsOur results indicated that vitamin C enhances the anti-proliferative and apoptotic effect of 5-AZA in HCC cell lines. By further analyzing the events leading to cell cycle arrest, we have shown for the first time in HCC that the combination of 5-AZA and vitamin C leads to an enhanced downregulation of Snail expression, a key transcription factor governing epithelial-mesenchymal transition (EMT) process, and cell cycle arrest.ConclusionsWe conclude that when combined with 5-AZA, vitamin C enhances TET activity in HCC cells, leading to induction of active demethylation. An increase in P21 expression as a consequence of downregulation of Snail accompanied by the induction of GADD45B expression is the main mechanism leading to cell cycle arrest in HCCs.

Highlights

Hepatocellular carcinoma (HCC) is the most common adult liver malignancy that shows relatively poor prognosis and rapid progression [1, 2]
While a very low release of Lactose dehydrogenase (LDH) was observed with 5-AZA and vitamin C individually, the combination of 5-AZA and vitamin C showed a high rate of cytotoxicity in both cell lines
In Huh7, a significant increase in the sub2N population was observed in cells treated with 5-AZA + vitamin C, with a slight increase in LDH compared to the 5-AZA single-treated cells (Fig. 1c)

Summary

Introduction

Hepatocellular carcinoma (HCC) is the most common adult liver malignancy that shows relatively poor prognosis and rapid progression [1, 2]. It is established that tumor cells undergo various epigenetic modifications, DNA hypermethylation, that could lead to an imbalance in regulation of pro- and anti-apoptotic genes, Recently, demethylation of 5-methylcytosine (5mC) to 5hydroxymethyl cytosine (5hmC) was shown to be mediated by ten-eleven translocation (TET) proteins [4,5,6]. Since hypermethylation of promoters of tumor suppressor genes has been identified as one of the principal factors supporting cancer development, demethylation agents have become the main focus of molecular-targeted therapeutics. Various in vitro studies have shown that 5azacytidine (5-AZA), a potent DNA methyl transferase inhibitor (DNMTi), leads to re-expression of silenced. Our lab clearly demonstrated that 5-AZA modulates the expression of genes through induction of TET2 and TET3, improving 5hmC generation in HCC and inhibiting cells proliferation [8]

Objectives

Methods

Results

Discussion

Conclusion