Induction of active demethylation and 5hmC formation by 5-azacytidine is TET2 dependent and suggests new treatment strategies against hepatocellular carcinoma

Abstract

Background: Global deregulation of DNA methylation is one of the crucial causes of hepato cellular carcinoma (HCC). It has been reported that the anti-cancer drug 5-azacytidine (5-AZA) mediates the activation of tumor suppressor genes through passive demethylation by inhibiting DNMT1. Recent evidence suggests that active demethylation which is mediated by ten-eleven translocation (TET) proteins may also be an important step to control global methylation. However, there exists a controversial discussion in which TET proteins are involved in the demethylation process in HCC. Therefore, we firstly wanted to identify which of the TETs are involved in demethylation and later to study whether or not 5-AZA could trigger the TET-dependent active demethylation process in HCC. HCC cell lines (Huh-7, HLE, HLF), primary human hepatocytes (hHeps), and tissues from both healthy (55 patients) and HCC patients (55 patients) were included in this study; mRNA levels of isocitrate dehydrogenase (IDH1, 2) and TETs (TET1-3) were studied via qPCR and confirmed by Western blot. The expression of 5hmC/5mC was determined by immunohistochemistry in human HCC tissues and the corresponding adjacent healthy liver. HCC cell lines were stimulated with 5-AZA (0-20 μM) and viability (Resazurin conversion), toxicity (LDH release), proliferation (PCNA), and 5hmC/5mC distribution were assessed. In addition, knockdown experiments on TET proteins in HCC cell lines using short interference RNAs (siRNAs), in the presence and absence of 5-AZA, were performed.

Results: Our data applying qPCR, immunofluorescence, and Western blotting clearly show that TET2 and TET3 but not TET1 were significantly decreased in HCC tissue and different HCC cell lines compared to non-tumor liver tissues and hHeps. In addition, we show here for the first time applying knockdown experiments that 5-AZA is able to trigger an active TET2-dependent demethylation process with concomitant significant changes in 5hmC/5mC in HCC cell lines and hHeps.

Conclusions: Our data clearly show that the expression and activity of TET2 and TET3 proteins but not TET1 are impaired in hepatocellular carcinoma leading to the reduction of 5hmC in HCCs. Furthermore, this study identified a novel function of 5-azacytidine in promoting a TET-mediated generation of 5hmC suggesting that the availability of 5-AZA in cancer cells will have various effects on different epigenetic targets. These findings may open new therapeutic strategies for epigenetic drugs to treat HCC.

Keywords: 5-Azacytidine; 5-Hydroxymethylcytosine; Cancer biomarker; DNA methylation; TET.

Fig. 1

Distribution of 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in HCC tissue. a IHC was performed using antibodies against 5hmC and 5mC in human normal and HCC tissues, the mean values of the IHC are shown (each group N = 55, n = 3). Four pictures are presented for each condition. The reduction of the 5hmC generation and an increase of 5mC in HCC samples compared with the corresponding normal liver samples were observed. Data are represented as box plot (whisker, Tukey). b IF staining was performed by using the antibody against 5hmC (red) for HCC cell lines (Huh7, HLE, and HLF). Cell nuclei were counterstained with Hoechst 33342 (blue). Scale bar are 20 μM. For quantification, 5hmC positive nuclei were counted. Statistical methods included paired t test and independent samples t test. All statistical tests were considered significant at α = 0.05 (p < 0.05)

Fig. 2

Reduction of 5hmC is associated with the substantial reduction of IDH1 and IDH2 and TET2 and TET3 gene expressions. a The expression of TET2, TET3, IDH1, and IDH2 decreased in HCC tissues in comparison to the matched normal liver. Real-time qRT-PCR was used to determine TETs and IDHs relative mRNA levels in HCCs while using β2M as an internal control for normalization. Data are expressed as fold change in mRNA expression compared to the normal liver control (indicated by dashed line at 1) The data are presented as box plot (N = 9, n = 3): center line represents the median, box limits represent the first and third quartiles, and whiskers represent 1.5 times the interquartile range (The p value were assessed by Student’s t test; bars show mean ± SE; p < 0.0001 for TET2, TET3, IDH1, and p = 0.0275 for IDH2). b The same pattern was confirmed by comparing HCC cell lines (Huh7, HLE, HLF) to primary human hepatocytes (N = 4, n = 3). (p value assed by one-way ANOVA; bars show mean ± SE) (p < 0.001)

Fig. 3

Distinct effect of 5-AZA on HCC cell lines viability, proliferation and apoptosis. a 5-AZA treatment reduced resazurin conversion in HCC cell lines (Huh7, HLE, and HLF). The mitochondria activity of cells decreased upon treatment with 5, 10, and 20 μM of 5-AZA for 24, 48, 96, and 120 h in time- and dose-dependent manner (N = 4, n = 3). b Twenty micromolars of 5-AZA inhibited the PCNA expression in HCC cell lines after 48 h of incubation. c Twenty micromolars of 5-AZA caused LDH release in HLE and HLF cells after 48 h; however, LDH released in culture supernants by Huh7 cells did not increase upon treatment 5-AZA after (N = 4, n = 3). d 5-AZA induced cell death in HLE and HLF cells. Determination of sub2N fractions as marker for apoptosis was measured after treatment of HCC cells with 20 μM 5-AZA for 48 h (p value assed by Student’s t test; bars show the mean ± SE of three independent experiments)

Fig. 4

5-AZA enhanced the generation of 5hmC in HCC cell lines. a Immunostaining showed that 20 μM of 5-AZA enhances the generation of 5hmC in HCC cell lines after 48 h. b The semi-quantitative analysis of immunofluorescence staining indicated that the incubation of HCC cells with 5-AZA for 48 h increased 5hmC level (p value assessed by Student’s t test; data are represented as mean ± SE)

Fig. 5

5-AZA enhanced the generation of 5hmC through induction of TETs expression in HCC cell lines. a Twenty micromolars of 5-AZA induced the expression of TET2 and TET3 in HCC cells after 48 h of incubation. The m-RNA level of TET2 and TET3 genes were determined by quantitative real-time PCR after 24 and 48 h (p value assed by one-way ANOVA; data represented as mean ± SE). b Expression of TET2 and TET3 was examined by Western blot analysis in HCC cell lines (Huh7, HLE, and HLF) after treatment with 20 μM 5-AZA for 24 and 48 h. c Twenty micromolars of 5-AZA did not influence the expression of IDH1 and IDH2 in HCC cells significantly after 48 h of incubation. Real-time qRT-PCR was used to determine TETs and IDHs relative mRNA levels in HCC cell lines while using GAPDH as an internal control for normalization

Fig. 6

The effect of 5-AZA is mediated by TET methylcytosine dioxygenase. a siRNAs targeting TET 2 and TET3 decreased the level of TET m-RNA to approximately 30 % for TET3 and 40 % for TET2 as compared with corresponding cells transfected cells with control siRNAs shown by quantitative RT-PCR (p value assed by Student’s t test; data are represented as mean ± SE). b Immunostaining shows that the knockdown down of TET genes expression in HCC cell lines, specifically TET2, decreased the basal level of 5hmC signal and attenuated the induction of 5hmC by 5-AZA (20 μM) treatment for 48 h