14 research outputs found
In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System
Accurate prediction of the potential hepatotoxic nature of new pharmaceuticals
remains highly challenging. Therefore, novel in vitro models with improved
external validity are needed to investigate hepatic metabolism and timely
identify any toxicity of drugs in humans. In this study, we examined the
effects of diclofenac, as a model substance with a known risk of
hepatotoxicity in vivo, in a dynamic multi-compartment bioreactor using
primary human liver cells. Biotransformation pathways of the drug and possible
effects on metabolic activities, morphology and cell transcriptome were
evaluated. Formation rates of diclofenac metabolites were relatively stable
over the application period of seven days in bioreactors exposed to 300 µM
diclofenac (300 µM bioreactors (300 µM BR)), while in bioreactors exposed to
1000 µM diclofenac (1000 µM BR) metabolite concentrations declined
drastically. The biochemical data showed a significant decrease in lactate
production and for the higher dose a significant increase in ammonia
secretion, indicating a dose-dependent effect of diclofenac application. The
microarray analyses performed revealed a stable hepatic phenotype of the cells
over time and the observed transcriptional changes were in line with
functional readouts of the system. In conclusion, the data highlight the
suitability of the bioreactor technology for studying the hepatotoxicity of
drugs in vitro
Age‑dependent concomitant changes in synaptic function and GABAergic pathway in the APP/PS1 mouse model
MELK-T1, a small-molecule inhibitor of protein kinase MELK, decreases DNA-damage tolerance in proliferating cancer cells
MELK-T1, a small-molecule inhibitor of protein kinase MELK, decreases DNA-damage tolerance in proliferating cancer cells
Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNA-mediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELK-T1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold.status: publishe
In Vitro Model for Hepatotoxicity Studies Based on Primary Human Hepatocyte Cultivation in a Perfused 3D Bioreactor System
MELK-T1, a small-molecule inhibitor of protein kinase MELK, decreases DNA-damage tolerance in proliferating cancer cells
Maternal embryonic leucine zipper kinase (MELK), a serine/threonine protein kinase, has oncogenic properties and is overexpressed in many cancer cells. The oncogenic function of MELK is attributed to its capacity to disable critical cell-cycle checkpoints and reduce replication stress. Most functional studies have relied on the use of siRNA/shRNAmediated gene silencing. In the present study, we have explored the biological function of MELK using MELK-T1, a novel and selective small-molecule inhibitor. Strikingly, MELK-T1 triggered a rapid and proteasome-dependent degradation of the MELK protein. Treatment of MCF-7 (Michigan Cancer Foundation-7) breast adenocarcinoma cells with MELKT1 induced the accumulation of stalled replication forks and double-strand breaks that culminated in a replicative senescence phenotype. This phenotype correlated with a rapid and long-lasting ataxia telangiectasia-mutated (ATM) activation and phosphorylation of checkpoint kinase 2 (CHK2). Furthermore, MELK-T1 induced a strong phosphorylation of p53 (cellular tumour antigen p53), a prolonged up-regulation of p21 (cyclin-dependent kinase inhibitor 1) and a down-regulation of FOXM1 (Forkhead Box M1) target genes. Our data indicate that MELK is a key stimulator of proliferation by its ability to increase the threshold for DNA-damage tolerance (DDT). Thus, targeting MELK by the inhibition of both its catalytic activity and its protein stability might sensitize tumours to DNA-damaging agents or radiation therapy by lowering the DNA-damage threshold
Integrating High-Dimensional Transcriptomics and Image Analysis Tools into Early Safety Screening: Proof of Concept for a New Early Drug Development Strategy
During
drug discovery and development, the early identification
of adverse effects is expected to reduce costly late-stage failures
of candidate drugs. As risk/safety assessment takes place rather late
during the development process and due to the limited ability of animal
models to predict the human situation, modern unbiased high-dimensional
biology readouts are sought, such as molecular signatures predictive
for <i>in vivo</i> response using high-throughput cell-based
assays. In this theoretical proof of concept, we provide findings
of an in-depth exploration of a single chemical core structure. Via
transcriptional profiling, we identified a subset of close analogues
that commonly downregulate multiple tubulin genes across cellular
contexts, suggesting possible spindle poison effects. Confirmation
via a qualified toxicity assay (<i>in vitro</i> micronucleus
test) and the identification of a characteristic aggregate-formation
phenotype via exploratory high-content imaging validated the initial
findings. SAR analysis triggered the synthesis of a new set of compounds
and allowed us to extend the series showing the genotoxic effect.
We demonstrate the potential to flag toxicity issues by utilizing
data from exploratory experiments that are typically generated for
target evaluation purposes during early drug discovery. We share our
thoughts on how this approach may be incorporated into drug development
strategies