Maria Luiza Silveira Mello

Valproic acid/sodium valproate (VPA) is a widely prescribed anticonvulsant and has also been used against certain tumor cells. It is a potent modulator of gene expression. Its ability to induce apoptosis has been well documented in HeLa cells. However, another form of cell death - mitotic catastrophe - has not yet been explored in VPA-treated HeLa cells. Here, we investigated the effects of VPA treatment on mitotic catastrophe characteristics, including morphological features and their frequencies, fluorescence intensity signals of caspase-2 and p53, and the expression and abundance of DNMT1 and DNMT3B. An increased frequency of mitotic catastrophe was observed not only morphologically, but also through enhanced induction of caspase-2, involvement of p53, at least under more drastic VPA treatment, but without a decrease in DNMT1 or DNMT3B levels. Additionally, enhancement of mitotic catastrophe coincided with a reduction in mitotic chromosome abnormalities. Increased DNMT3B expression following VPA action, may be favored by previously reported chromatin decondensation induced by this drug. Enhanced CpG methylation of specific DNA sites could thus be promoted. In conclusion, VPA was shown to trigger metabolic pathways linked to different forms of cell death in HeLa cells, supporting its oncosuppressive potential.

Tenocytes migrating from cultured rat tendon explants aggregate and gradually align in parallel rows with the tendon’s long axis as the explant period extends up to 12 days and collagen bundles become structurally remodeled. Tendon architecture and mechanical load are known to affect fibroblast phenotypes; thus, we studied cell cultures derived from tenocytes at early and later migration stages using image analysis and immunofluorescence for selected epigenetic markers to identify differences in nuclear characteristics across migration periods. Feulgen-DNA amounts and global DNA 5-methylcytosine and acetylated H3K9 distribution were not found to differ in the cells originating from tenocytes that migrated at the 8- and 12-day periods of tendon culture. However, light absorbance, fractal dimension, and margination features related to chromatin condensation and distribution in the Feulgen-stained cell nuclei differed during these periods. Accumulation of the H3K4me2 marker at the nuclear periphery in cells obtained after a longer explant culture period, when the margination feature reaches larger values, may represent the acquisition of an enhanced transcriptional memory that could affect future gene expression levels. Further assays are required to determine whether the present image analysis results correlate with the differential potential of these cells for extracellular matrix synthesis and practical bioengineering purposes.

The development and progression of glioblastoma, the most aggressive malignant intracranial tumor with a poor prognosis, are influenced by mutations, the overexpression of oncogenes, and epigenetic factors, particularly those related to DNA methylation status and histone post-translational modifications. Valproic acid (VPA), a classic histone deacetylase (HDAC) inhibitor, has shown promise both on its own and in combination with other drugs as a therapeutic agent against various solid tumors, including gliomas. Given VPA’s reported effects on chromatin supraorganization and expression activity in several cell types, we studied textural features that may indicate changes in chromatin structure in U-251MG glioblastoma cells cultured in the presence of VPA, utilizing image cytometry. For comparison, cells treated with 5-aza-CdR served as a positive control for DNA demethylation. Chromatin remodeling was observed in VPA-treated cells, which displayed decreased HDAC activity and increased histone H3 acetylation, whereas no such changes were detected in 5-aza-CdR-treated cells. These findings suggest that, despite the significance of DNA methylation alterations in glioblastoma cells, the chromatin remodeling observed through image cytometry in VPA-treated U-251MG cells is more closely associated with induced changes involving histone modifications rather than with DNA demethylation.

P16^INK4a and p21^WAF1/Cip1 are cyclin‑dependent kinase inhibitors involved in cell cycle control, which can function as oncogenes or tumor suppressors, depending on the context of various extracellular and intracellular signals, and cell type. In human papillomavirus‑induced cervical cancer, p16^INK4a shows oncogenic activity and functions as a diagnostic marker of cervical neoplasia, whereas p21^WAF1/Cip1 acts as a tumor suppressor and its downregulation is associated with the progression of malignant transformation. Several histone deacetylase (HDAC) inhibitors promote the positive and negative regulation of a number of genes, including p16^INK4a and p21^WAF1/Cip1; however, the effects of sodium valproate (VPA) on these genes and on the proteins they encode remain uncertain in HeLa cervical cancer cells. In the present study, these effects were investigated in HeLa cells treated with 0.5 or 2 mM VPA for 24 h, using reverse transcription‑quantitative PCR, confocal microscopy and western blotting. The results revealed a decrease in the mRNA expression levels of p16^INK4a and a tendency for p16^INK4a protein abundance to decrease in the presence of 2 mM VPA. By contrast, an increase in the protein expression levels of p21^WAF1/Cip1 was detected in the presence of 0.5 and 2 mM VPA. Furthermore, VPA was confirmed to inhibit HDAC activity and induce global hyperacetylation of histone H3. Notably, VPA was shown to suppress p16^INK4a, a biomarker gene of cervical carcinoma, and to increase the abundance of the tumor suppressor protein p21^WAF1/Cip1, thus contributing to the basic knowledge regarding the antitumorigenic potential of VPA. Exploration of epigenetic changes associated with the promoters of p16^INK4a and p21^WAF1/Cip1, such as histone H3 methylation, may provide further information and improve the understanding of these findings.

Valproic acid in association with sodium valproate (VPA) is an important anticonvulsant drug used for decades to treat neurological disorders. VPA also acts as an epigenetic modulator by inhibiting histone deacetylases, permitting histone acetylation, affecting the DNA and histone methylation status and gene expression, and inducing chromatin remodeling. Insects represent an important animal model for studies in several areas of science. Their high phenotypic plasticity makes them alternative models for epigenetic studies. This brief review emphasizes recent reports on insect epigenetics and the contribution of studies on the VPA action in insects, including effects on epigenetic markers, extending the pharmacological understanding of the potential of this drug, and demonstrating the usefulness of insects as an alternative animal model to drug studies.

Sodium valproate (VPA) is an epidrug that inhibits histone deacetylases and favors histone acetylation and DNA demethylation, thereby inducing chromatin remodeling in several cell types. Although VPA induced heterochromatin decondensation in the hemipteran Triatoma infestans, this phenomenon was not favored by histone acetylation. Considering that DNA demethylation leading to chromatin unpacking is another consequence of VPA action in several organisms, we used immunofluorescence to investigate whether cytosine methylation was the target of VPA action that affected heterochromatin remodeling in T. infestans. In addition, Fourier transform infrared (FTIR) analysis was used to evaluate the participation of cytosine in T. infestans DNA. Immunofluorescence results indicated the absence of 5-methylcytosine signals in heterochromatin, but the presence of these signals in euchromatin, where demethylation was promoted by VPA. The FTIR spectra of T. infestans global DNA exhibited marks indicative of AT richness and a low representation of cytosine contribution. In conclusion, the chromatin remodeling event previously reported in the heterochromatin compartment of T. infestans in response to VPA, which was not due to histone acetylation, was also not due to DNA cytosine demethylation.

Triatoma infestans (Klug) is an insect recognized as not only an important vector of South American trypanosomiasis (Chagas disease) but also a model of specific cellular morphofunctional organization and epigenetic characteristics. The purpose of the present review is to highlight certain cellular processes that are particularly unveiled in T. infestans, such as the following: (1) somatic polyploidy involving nuclear and cell fusions that generate giant nuclei; (2) diversification of nuclear phenotypes in the Malpighian tubules during insect development; (3) heterochromatin compartmentalization into large bodies with specific spatial distribution and presumed mobility in the cell nuclei; (4) chromatin remodeling and co-occurrence of necrosis and apoptosis in the Malpighian tubules under stress conditions; (5) epigenetic markers; and (6) response of heterochromatin to valproic acid, an epidrug that inhibits histone deacetylases and induces DNA demethylation in other cell systems. These cellular processes and epigenetic characteristics emphasize the role of T. infestans as an attractive model for cellular research. A limitation of these studies is the availability of insect supply by accredited insectaries. For studies that require the injection of drugs, the operator’s dexterity to perform insect manipulation is necessary, especially if young nymphs are used. For studies involving in vitro cultivation of insect organs, the culture medium should be carefully selected to avoid inconsistent results.