DNA Methylation Changes in Valproic Acid-Treated HeLa Cells as Assessed by Image Analysis, Immunofluorescence and Vibrational Microspectroscopy

Abstract

Valproic acid (VPA), a well-known histone deacetylase inhibitor, has been reported to affect the DNA methylation status in addition to inducing histone hyperacetylation in several cell types. In HeLa cells, VPA promotes histone acetylation and chromatin remodeling. However, DNA demethylation was not checked in this cell model for standing effects longer than those provided by histone acetylation, which is a rapid and transient phenomenon. Demonstration of VPA-induced DNA demethylation in HeLa cells would contribute to understanding the effect of VPA on an aggressive tumor cell line. In the present work, DNA demethylation in VPA-treated HeLa cells was assessed by image analysis of chromatin texture, the abundance of 5-methylcytosine (5mC) immunofluorescence signals and Fourier transform-infrared (FT-IR) microspectroscopy centered on spectral regions related to the vibration of–CH3 groups. Image analysis indicated that increased chromatin unpacking promoted by a 4-h-treatment with 1.0 mM VPA persisted for 24 h in the absence of the drug, suggesting the occurrence of DNA demethylation that was confirmed by decreased 5mC immunofluorescence signals. FT-IR spectra of DNA samples from 1 mM or 20 mM VPA-treated cells subjected to a peak fitting analysis of the spectral window for–CH3 stretching vibrations showed decreased vibrations and energy of these groups as a function of the decreased abundance of 5mC induced by increased VPA concentrations. Only the 20 mM-VPA treatment caused an increase in the ratio of -CH3 bending vibrations evaluated at 1375 cm-1 in relation to in-plane vibrations of overall cytosines evaluated at 1492 cm-1. CH3 stretching vibrations showed to be more sensitive than–CH3 bending vibrations, as detected with FT-IR microspectroscopy, for studies aiming to associate vibrational spectroscopy and changes in DNA 5mC abundance.

Cellular Effects of the Antiepileptic Drug Valproic Acid in Glioblastoma

“…In addition, VPA at a concentration of 750 µM did not radiosensitize the three human glioblastoma cell lines and three primary glioblastoma spheroid cultures studied in delayed plating colony formation assays and pre-plating limited dilution assays, respectively. These results were unexpected in light of the reported chromatin remodeling action of VPA [18, 48-50] and contradictory to previous in vitro studies [10-15, 17, 48, 51, 52] which clearly show VPA-induced cell death or radio- or chemosensitization in cancer cells including glioma. Unlike the present work, VPA concentrations of 1-5 mM [52], 1.5-2 mM [10], 1-5 mM [51], 2.5-5 mM [11], 1.5-3 mM [12], 1-15 mM [13], 2-16 mM [14], 7.5 mM [18], 2 mM [19], 5-20 mM [53], 1-10 mM [21], or 4.8 mM [22] were applied in these studies.…”

Exploring the Drug Repurposing Versatility of Valproic Acid as a Multifunctional Regulator of Innate and Adaptive Immune Cells

“…Conversely, the ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, promoting the reversal of DNA methylation and gene silencing [31]. VPA also decreases methylated DNA in different cell lines, including neuroblastoma, HEK 293, and HeLa [32–34]. In addition, VPA-treated mice showed increased DNA demethylation in their brains [35].…”

Embryo‐derived teratoma in vitro biological system reveals antitumor and embryotoxic activity of valproate

“…FTIR spectroscopy is detecting vibrations of chemical and functional groups of molecules within complex biological samples to obtain a spectral fingerprint and was proposed for the fast analysis of biofluids, allowing the subsequent classification of spectra from different categories with computational methods and possibly the identification of biomarkers [47]. The advantages of this label-free approach are the fast analysis of a large number of samples from minimum material, and a low cost [47][48][49][50].…”