Valproic acid alters the content and function of the cell-free DNA released by hepatocellular carcinoma (HepG2) cells in vitro.

BACKGROUND: It has long been believed that cell-free DNA (cfDNA) actively released into circulation can serve as intercellular messengers, and their involvement in processes such as the bystander effect strongly support this. However, this intercellular messaging function of cfDNA may have clinical implications that have not yet been considered. METHODS: CfDNA was isolated from the growth medium of HepG2 cells treated with valproic acid (VPA). This cfDNA was then administered to untreated cells and cellular metabolic activity was measured. RESULTS: VPA altered the characteristics of cfDNA released by treated HepG2 cells in vitro. When administered to untreated cells, the cfDNA from cells treated with VPA resulted in the dose-dependent induction of glycolytic activity within 36 min of administration, but little to no alterations in oxidative phosphorylation. The glycolytic activity lasted for 4-6 h, whereas changes in subsequent cfDNA release and characteristics were found to remain persistent after two 24 h treatments. Fragmented genomic DNA from VPA-treated cells did not induce the effects observed for cfDNA obtained VPA-treated cells. CONCLUSIONS: It is possible for cfDNA to, under in vitro conditions, transfer pharmaceutically-induced effects to untreated recipient cells. Further investigation regarding this occurrence under in vivo conditions is, therefore, strongly encouraged. GENERAL SIGNIFICANCE: The intercellular messaging functions of cfDNA present in donated biological fluids has potential clinical implications that require urgent attention. These implications may, however, also have potential as new forms of treatment that can circumvent pharmacological barriers.

Kinetic analysis, size profiling, and bioenergetic association of DNA released by selected cell lines in vitro.

Although circulating DNA (cirDNA) analysis shows great promise as a screening tool for a wide range of pathologies, numerous stumbling blocks hinder the rapid translation of research to clinical practice. This is related directly to the inherent complexity of the in vivo setting, wherein the influence of complex systems of interconnected cellular responses and putative DNA sources creates a seemingly arbitrary representation of the quantitative and qualitative properties of the cirDNA in the blood of any individual. Therefore, to evaluate the potential of in vitro cell cultures to circumvent the difficulties encountered in in vivo investigations, the purpose of this work was to elucidate the characteristics of the DNA released [cell-free DNA (cfDNA)] by eight different cell lines. This revealed three different forms of cfDNA release patterns and the presence of nucleosomal fragments as well as actively released forms of DNA, which are not only consistently observed in every tested cell line, but also in plasma samples. Correlations between cfDNA release and cellular origin, growth rate, and cancer status were also investigated by screening and comparing bioenergetics flux parameters. These results show statistically significant correlations between cfDNA levels and glycolysis, while no correlations between cfDNA levels and oxidative phosphorylation were observed. Furthermore, several correlations between growth rate, cancer status, and dependency on aerobic glycolysis were observed. Cell cultures can, therefore, successfully serve as closed-circuit models to either replace or be used in conjunction with biofluid samples, which will enable sharper focus on specific cell types or DNA origins.

Curcumin Rescues a PINK1 Knock Down SH-SY5Y Cellular Model of Parkinson's Disease from Mitochondrial Dysfunction and Cell Death.

Parkinson's disease (PD) is a neurodegenerative disorder characterised by the loss of dopaminergic neurons in the substantia nigra. Mutations in the PINK1 gene result in an autosomal recessive form of early-onset PD. PINK1 plays a vital role in mitochondrial quality control via the removal of dysfunctional mitochondria. The aim of the present study was to create a cellular model of PD using siRNA-mediated knock down of PINK1 in SH-SY5Y neuroblastoma cells The possible protective effects of curcumin, known for its many beneficial properties including antioxidant and anti-inflammatory effects, was tested on this model in the presence and absence of paraquat, an additional stressor. PINK1 siRNA and control cells were separated into four treatment groups: (i) untreated, (ii) treated with paraquat, (iii) pre-treated with curcumin then treated with paraquat, or (iv) treated with curcumin. Various parameters of cellular and mitochondrial function were then measured. The PINK1 siRNA cells exhibited significantly decreased cell viability, mitochondrial membrane potential (MMP), mitochondrial respiration and ATP production, and increased apoptosis. Paraquat-treated cells exhibited decreased cell viability, increased apoptosis, a more fragmented mitochondrial network and decreased MMP. Curcumin pre-treatment followed by paraquat exposure rescued cell viability and increased MMP and mitochondrial respiration in control cells, and significantly decreased apoptosis and increased MMP and maximal respiration in PINK1 siRNA cells. These results highlight a protective effect of curcumin against mitochondrial dysfunction and apoptosis in PINK1-deficient and paraquat-exposed cells. More studies are warranted to further elucidate the potential neuroprotective properties of curcumin.

Altered Mitochondrial Respiration and Other Features of Mitochondrial Function in Parkin-Mutant Fibroblasts from Parkinson's Disease Patients.

Mutations in the parkin gene are the most common cause of early-onset Parkinson's disease (PD). Parkin, an E3 ubiquitin ligase, is involved in respiratory chain function, mitophagy, and mitochondrial dynamics. Human cellular models with parkin null mutations are particularly valuable for investigating the mitochondrial functions of parkin. However, published results reporting on patient-derived parkin-mutant fibroblasts have been inconsistent. This study aimed to functionally compare parkin-mutant fibroblasts from PD patients with wild-type control fibroblasts using a variety of assays to gain a better understanding of the role of mitochondrial dysfunction in PD. To this end, dermal fibroblasts were obtained from three PD patients with homozygous whole exon deletions in parkin and three unaffected controls. Assays of mitochondrial respiration, mitochondrial network integrity, mitochondrial membrane potential, and cell growth were performed as informative markers of mitochondrial function. Surprisingly, it was found that mitochondrial respiratory rates were markedly higher in the parkin-mutant fibroblasts compared to control fibroblasts (p = 0.0093), while exhibiting more fragmented mitochondrial networks (p = 0.0304). Moreover, cell growth of the parkin-mutant fibroblasts was significantly higher than that of controls (p = 0.0001). These unanticipated findings are suggestive of a compensatory mechanism to preserve mitochondrial function and quality control in the absence of parkin in fibroblasts, which warrants further investigation.