Neuronal life or death linked to depression treatment: the interplay between drugs and their stress-related outcomes relate to single or combined drug therapies.

Depression is a serious medical condition, typically treated by antidepressants. Conventional monotherapy can be effective only in 60-80% of patients, thus modern psychiatry deals with the challenge of new methods development. At the same moment, interactions between antidepressants and the occurrence of potential side effects raise serious concerns, which are even more exacerbated by the lack of relevant data on exact molecular mechanisms. Therefore, the aims of the study were to provide up-to-date information on the relative mechanisms of action of single antidepressants and their combinations. In this study, we evaluated the effect of single and combined antidepressants administration on mouse hippocampal neurons after 48 and 96 h in terms of cellular and biochemical features in vitro. We show for the first time that co-treatment with amitriptyline/imipramine + fluoxetine initiates in cells adaptation mechanisms which allow cells to adjust to stress and finally exerts less toxic events than in cells treated with single antidepressants. Antidepressants treatment induces in neuronal cells oxidative and nitrosative stress, which leads to micronuclei and double-strand DNA brakes formation. At this point, two different mechanistic events are initiated in cells treated with single and combined antidepressants. Single antidepressants (amitriptyline, imipramine or fluoxetine) activate cell cycle arrest resulting in proliferation inhibition. On the other hand, treatment with combined antidepressants (amitriptyline/imipramine + fluoxetine) initiates p16-dependent cell cycle arrest, overexpression of telomere maintenance proteins and finally restoration of proliferation. In conclusion, our findings may pave the way to better understanding of the stress-related effects on neurons associated with mono- and combined therapy with antidepressants.

Publishing interim results of randomised clinical trials in peer-reviewed journals.

BACKGROUND: Interim analyses of randomised controlled trials are sometimes published before the final results are available. In several cases, the treatment effects were noticeably different after patient recruitment and follow-up completed. We therefore conducted a literature review of peer-reviewed journals to compare the reported treatment effects between interim and final publications and to examine the magnitude of the difference. METHODS: We performed an electronic search of MEDLINE from 1990 to 2014 (keywords: 'clinical trial' OR 'clinical study' AND 'random*' AND 'interim' OR 'preliminary'), and we manually identified the corresponding final publication. Where the electronic search produced a final report in which the abstract cited interim results, we found the interim publication. We also manually searched every randomised controlled trial in eight journals, covering a range of impact factors and general medical and specialist publications (1996-2014). All paired articles were checked to ensure that the same comparison between interventions was available in both. RESULTS: In all, 63 studies are included in our review, and the same quantitative comparison was available in 58 of these. The final treatment effects were smaller than the interim ones in 39 (67%) trials and the same size or larger in 19 (33%). There was a marked reduction, defined as a ≥20% decrease in the size of the treatment effect from interim to final analysis, in 11 (19%) trials compared to a marked increase in 3 (5%), p = 0.057. The magnitude of percentage change was larger in trials where commercial support was reported, and increased as the proportion of final events at the interim report decreased in trials where commercial support was reported (interaction p = 0.023). There was no evidence of a difference between trials that stopped recruitment at the interim analysis where this was reported as being pre-specified versus those that were not pre-specified (interaction p = 0.87). CONCLUSION: Published interim trial results were more likely to be associated with larger treatment effects than those based on the final report. Publishing interim results should be discouraged, in order to have reliable estimates of treatment effects for clinical decision-making, regulatory authority reviews and health economic analyses. Our work should be expanded to include conference publications and manual searches of additional journal publications.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Synergetic effects of DNA demethylation and histone deacetylase inhibition in primary rat hepatocytes.

Both, DNA methylation and histone deacetylation play a crucial role in cancer development by silencing the expression of specific tumour suppressor genes. Several studies describe the use of combinations of DNA methyltransferase inhibitors (DNMT-i) and histone deacetylase inhibitors (HDAC-i) as an improved strategy to treat neoplasms. However, no information is available concerning their biological impact on healthy, non-malignant cells, including hepatocytes. Therefore, the effects of the combination of the DNMT-i decitabine (DAC) with the HDAC-i 6-[(4-pyrrolidine-1-ylbenzoyl) amino] hexanoic acid hydroxamate (AN-8) on cell proliferation and differentiation were examined in primary rat hepatocyte cultures. We found that, upon simultaneous exposure of the cells to both compounds, a synergetic anti-proliferative outcome was achieved. This inhibition of DNA synthesis was accompanied by a reduced expression of cyclin-dependent kinase 1 (cdk1), a key cell cycle marker that controls the S/G2/M transition. Compared to exposure of the cells to each agent separately, the combination of lower concentrations of both DAC and AN-8 promoted the maintenance of the differentiated phenotype of the cells as a function of culture time. The functionality of the hepatocytes was evidenced by an increased expression of the phase I biotransformation enzyme cytochrome P 450 (CYP) 1A1 and albumin secretion capacity when both agents were used in combination.

Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation.

Controlling both growth and differentiation of stem cells and their differentiated somatic progeny is a challenge in numerous fields, from preclinical drug development to clinical therapy. Recently, new insights into the underlying molecular mechanisms have unveiled key regulatory roles of epigenetic marks driving cellular pluripotency, differentiation and self-renewal/proliferation. Indeed, the transcription of genes, governing cell-fate decisions during development and maintenance of a cell's differentiated status in adult life, critically depends on the chromatin accessibility of transcription factors to genomic regulatory and coding regions. In this review, we discuss the epigenetic control of (liver-specific) gene-transcription and the intricate interplay between chromatin modulation, including histone (de)acetylation and DNA (de)methylation, and liver-enriched transcription factors. Special attention is paid to their role in directing hepatic differentiation of primary hepatocytes and stem cells in vitro.

Biology and pathobiology of gap junctional channels in hepatocytes.

The present review provides the state of the art of the current knowledge concerning gap junctional channels and their roles in liver functioning. In the first part, we summarize some relevant biochemical properties of hepatic gap junctional channels, including their structure and regulation. In the second part, we discuss the involvement of gap junctional channels in the occurrence of liver cell growth, liver cell differentiation, and liver cell death. We further exemplify their relevance in hepatic pathophysiology. Finally, a number of directions for future liver gap junctional channel research are proposed, and the up-regulation of gap junctional channel activity as a novel strategy in (liver) cancer therapy is illustrated.

Screening of amide analogues of Trichostatin A in cultures of primary rat hepatocytes: search for potent and safe HDAC inhibitors.

The vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug-target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes.

Epigenetic Modifications as Antidedifferentiation Strategy for Primary Hepatocytes in Culture.

A well-known problem of cultured primary hepatocytes is their rapid dedifferentiation. During the last years, several strategies to counteract this phenomenon have been developed, of which changing the in vitro environment is the most popular one. However, mimicking the in vivo setting in vitro by adding soluble media additives or the restoration of both cell-cell and cell-extracellular matrix contacts is not sufficient and only delays the dedifferentiation process instead of counteracting it. In this chapter, new strategies to prevent the deterioration of the liver-specific phenotype of primary hepatocytes in culture by targeting the (epi)genetic mechanisms that drive hepatocellular gene expression are described.

Toxicological and metabolic considerations for histone deacetylase inhibitors.

INTRODUCTION: Vorinostat and romidepsin were the first histone deacetylase (HDAC) inhibitors (HDi) that fulfilled the preclinical promise of anticancer potential in clinical trials. Nevertheless, they merely opened a new chapter in the history of cancer therapy. Demonstration of their antitumor activity was a straightforward task in in vitro setting. Proving their efficacy in vivo was much more difficult, since the effects of an administrated drug strongly depend on its absorption, distribution, metabolism and excretion. AREAS COVERED: This article summarizes clinical data on the pharmacokinetic properties of HDi that are currently at more advanced stages of clinical development. Specific attention is paid to the metabolic pathways. Moreover, a comprehensive overview of HDi-related adverse effects is given. EXPERT OPINION: At this moment, HDi form one of the most interesting classes of therapeutics, yet their efficacy and safety profiles could still be improved by i) designing better formulations, ii) more extensive characterization of their disposition at the preclinical stage, iii) targeting of individual disease-related deacetylase isoforms and/or their complexes, iv) selecting a target patient population with the highest probability of response based on molecular signatures.

Effect of Trichostatin A on miRNA expression in cultures of primary rat hepatocytes.

In the present study, the effect of Trichostatin A (TSA), a histone deacetylase inhibitor, was investigated on the microRNA (miR, miRNA) expression profile in cultured primary rat hepatocytes by means of microarray analysis. Simultaneously, albumin secretory capacity and morphological features of the hepatocytes were evaluated throughout the culture time. In total, 25 out of 348 miRNAs were found to be differentially expressed between freshly isolated hepatocytes and 7-day cultured cells. Nineteen of these miRNAs were connected with 'general metabolism'. miR-21 and miR-126 were shown to be the most up and down regulated miRs upon cultivation and could be linked to the proliferative response triggered in the hepatocytes upon their isolation from the liver. miR-379 and miR-143, on the other hand, were found to be the most up and down regulated miRs upon TSA treatment. Together with the higher expression of miR-122 observed in TSA-treated versus non-treated cultures, we hypothesize that the changes observed for miR-122, miR-143 and miR-379 could be related to the inhibitory effects of TSA on hepatocellular proliferation.

Preservation of hepatocellular functionality in cultures of primary rat hepatocytes upon exposure to 4-Me2N-BAVAH, a hydroxamate-based HDAC-inhibitor.

Great efforts are being put in the development/optimization of reliable and highly predictive models for high-throughput screening of efficacy and toxicity of promising drug candidates. The use of primary hepatocyte cultures, however, is still limited by the occurrence of phenotypic alterations, including loss of xenobiotic biotransformation capacity. In the present study, the differentiation-stabilizing effect of a new histone deacetylase inhibitor 5-(4-dimethylaminobenzoyl)-aminovaleric acid hydroxamide (4-Me(2)N-BAVAH), a structural Trichostatin A (TSA)-analogue with a more favourable pharmaco-toxicological profile, was studied at a genome-wide scale by means of microarray analysis. Several genes coding for xenobiotic biotransformation enzymes were found to be positively regulated upon exposure to 4-Me(2)N-BAVAH. For CYP1A1/2B1/3A2, these observations were confirmed by qRT-PCR and immunoblot analysis. In addition, significantly higher 7-ethoxyresorufin-O-deethylase and 7-pentoxyresorufin-O-dealkylase activity levels were measured. These effects were accompanied by an increased expression of CCAAT/enhancer binding protein alpha and hepatic nuclear factor (HNF)4α, but not of HNF1α. Finally, 4-Me(2)N-BAVAH was found to induce histone H3 acetylation at the proximal promoter of the albumin, CYP1A1 and CYP2B1 genes, suggesting that chromatin remodelling is directly involved in the transcriptional regulation of these genes. In conclusion, histone deacetylase inhibitors prove to be efficient agents for better maintaining a differentiated hepatic phenotype in rat hepatocyte cultures.

DNA methyltransferase 3a expression decreases during apoptosis in primary cultures of hepatocytes.

Although it has been acknowledged that DNA methylation is a key factor in the epigenetic control of liver homeostasis, its role in the occurrence of hepatocyte cell death has yet been poorly documented. We therefore have investigated the expression pattern of the effectors of DNA methylation, namely DNA methyltransferase (DNMT) isoenzymes, during Fas-mediated apoptotic cell death in primary hepatocyte cultures. Cell death was assessed by in situ stainings with Annexin V, Hoechst 33342 and Propidium iodide, and measurement of caspase 3-like activity and lactate dehydrogenase release. Similar to the hepatic in vivo situation, DNMT1, DNMT2 and DNMT3b could not be detected, whereas relatively high levels of DNMT3a protein were observed in the in vitro setting, as studied by immunoblotting. Upon induction of cell death, a progressive decrease in DNMT3a protein amount was noticed, reaching a minimum level towards the final stages of the cell death process. This was preceded by parallel changes in DNMT3a mRNA production, measured by qRT-PCR analysis, which became already evident during the early stages of apoptosis. We conclude that downregulated DNMT3a protein production during Fas-mediated hepatocyte apoptosis results from inhibition of DNMT3a gene transcription. This finding further substantiates the existence of an epigenetic signature of apoptosis.

Regulated phosphorylation of 40S ribosomal protein S6 in root tips of maize.

Ribosomal protein S6 (RPS6) is located in the mRNA binding site of the 40S subunit of cytosolic ribosomes. Two maize (Zea mays) rps6 genes were identified that encode polypeptides (30 kD, 11.4 pI) with strong primary amino acid sequence and predicted secondary structure similarity to RPS6 of other eukaryotes. Maize RPS6 was analyzed by the use of two-dimensional gel electrophoresis systems, in vivo labeling with [(32)P]P(i) and immunological detection. Nine RPS6 isoforms were resolved in a two-dimensional basic-urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry performed on trypsin-digested isoforms identified four serine (Ser) and one threonine (Thr) residue in the carboxy-terminal region as phosphorylation sites (RRS(238)KLS(241)AAAKAS(247)AAT(250)S(251)A-COOH). Heterogeneity in RPS6 phosphorylation was a consequence of the presence of zero to five phosphorylated residues. Phosphorylated isoforms fell into two groups characterized by (a) sequential phosphorylation of Ser-238 and Ser-241 and (b) the absence of phospho-Ser-238 and presence of phospho-Ser-241. The accumulation of hyper-phosphorylated isoforms with phospho-Ser-238 was reduced in response to oxygen deprivation and heat shock, whereas accumulation of these isoforms was elevated by cold stress. Salt and osmotic stress had no reproducible effect on RPS6 phosphorylation. The reduction in hyper-phosphorylated isoforms under oxygen deprivation was blocked by okadaic acid, a Ser/Thr phosphatase inhibitor. By contrast, the recovery of hyper-phosphorylated isoforms upon re-oxygenation was blocked by LY-294002, an inhibitor of phosphatidylinositol 3-kinases. Thus, differential activity of phosphatase(s) and kinase(s) determine complex heterogeneity in RPS6 phosphorylation.