Sodium valproate and 5-aza-2′-deoxycytidine differentially modulate DNA demethylation in G1 phase-arrested and proliferative HeLa cells

Abstract

Sodium valproate/valproic acid (VPA), a histone deacetylase inhibitor, and 5-aza-2-deoxycytidine (5-aza-CdR), a DNA methyltransferase 1 (DNMT1) inhibitor, induce DNA demethylation in several cell types. In HeLa cells, although VPA leads to decreased DNA 5-methylcytosine (5mC) levels, the demethylation pathway involved in this effect is not fully understood. We investigated this process using flow cytometry, ELISA, immunocytochemistry, Western blotting and RT-qPCR in G1 phasearrested and proliferative HeLa cells compared to the presumably passive demethylation promoted by 5-aza-CdR. The results revealed that VPA acts predominantly on active DNA demethylation because it induced TET2 gene and protein overexpression, decreased 5mC abundance, and increased 5-hydroxymethylcytosine (5hmC) abundance, in both G1-arrested and proliferative cells. However, because VPA caused decreased DNMT1 gene expression levels, it may also act on the passive demethylation pathway. 5-aza-CdR attenuated DNMT1 gene expression levels but increased TET2 and 5hmC abundance in replicating cells, although it did not affect the gene expression of TETs at any stage of the cell cycle. Therefore, 5-aza-CdR may also function in the active pathway. Because VPA reduces DNA methylation levels in non-replicating HeLa cells, it could be tested as a candidate for the therapeutic reversal of DNA methylation in cells in which cell division is arrested.

Sodium Valproate-Induced Chromatin Remodeling

“…Valproic acid has also been found to induce chromatin decondensation that lasts longer than the time assigned to promote histone acetylation. This finding suggested that VPA could affect the methylation status of DNA and histones, which was confirmed in several cell types, including tumor cells (Detich et al, 2003;Milutinovic et al, 2007;Marinova et al, 2011;Palsamy et al, 2014;Rocha et al, 2019). The VPA-promoted demethylation of DNA, which leads to the conversion of 5-methylcytosine (5mC) to cytosine (C), involves a complex process flowing through an active or a passive pathway, depending on the cell type.…”

“…In MCF-7 human breast tumor cells, for instance, VPA-induced DNA demethylation occurs through a passive pathway (Marchion et al, 2005). In human lens epithelial cells and HeLa cells, VPA acts predominantly within the active DNA demethylation pathway, through the action of enzymes of the ten-eleven translocation (TET) protein family and independent of cell replication, although a passive pathway promoting the suppression of DNA methyltransferase (DNMT) activity may also be involved (Palsamy et al, 2014;Rocha et al, 2019). Although reversible, DNA methylation changes are more stable than histone acetylation alterations, and may lead to long-term epigenetic reprogramming (Milutinovic et al, 2007).…”

Valproic acid influences the expression of genes implicated with hyperglycaemia-induced complement and coagulation pathways

“…The experimental results presented in this article suggests lysine acetylation is associated with VPA attenuating MASP and C3 gene expression we cannot rule out histone deacetylation of other complement genes. Furthermore, in addition to causing histone hyperacetylation, VPA has been shown to regulate replication-independent loss of DNA methylation 72 that is consistent with elevated Tet2 DNA demethylase enzyme 73 . This seemingly extends the functional role of HDAC inhibitors such as VPA that alter lysine acetylation and deacetylation including DNA methylation by extracellular signalling mediated by hyperglycaemia 74 .…”

Cancer and Tumour Suppressor p53 Encounters at the Juncture of Sex Disparity

“…These are proposed tumour suppressor genes: with PGP9.5 able to bind and stabilise p53 by preventing its proteasomal degradation; and NMDAR2B and CCNA1 acting to facilitate apoptosis ( Waraya et al, 2015 ). The therapeutic importance of these findings is that epigenetic modifiers able to reduce methylation [e.g., 5-aza-2’-deoxycytidine (5-aza-dC), a DNA methyltransferase 1 (DNMT1) inhibitor]; in combination with sodium valproate or valproic acid [a histone deacetylase inhibitor ( Rocha et al, 2019 )], will have specific value, with greater relevant application in these male gastric cancers, due to their higher methylation incidence. Altered methylation (either 11 hyper-methylated and seven hypomethylated) of ∼30% of genes (18/67) in the p53 KEGG pathway were also found in colorectal cancer ( Molnar et al, 2018 ), which now begs for interrogation of sex differences.…”