Assessment of Mitochondrial Cell Metabolism by Respiratory Chain Electron Flow Assays

in Weissig, Volkmar; Edeas, Marvin (Eds.) Methods in Molecular Biology (2021)

Cellular energy metabolism is regulated by complex metabolic pathways. Although anaerobic glycolysis was reported as a primary source of energy in cancer leading to a high rate of lactate production ... [more ▼]

Cellular energy metabolism is regulated by complex metabolic pathways. Although anaerobic glycolysis was reported as a primary source of energy in cancer leading to a high rate of lactate production, current evidence shows that the main energy source supporting cancer cell metabolism relies on mitochondrial metabolism. Mitochondria are the key organelle maintaining optimal cellular energy levels. MitoPlate™ S-1 provides a highly reproducible bioenergetics tool to analyze the electron flow rate in live cells. Measuring the rates of electron flow into and through the electron transport chain using different NADH and FADH2-producing metabolic substrates enables the assessment of mitochondrial functionality. MitoPlate™ S-1 are 96-well microplates pre-coated with different substrates used as probes to examine the activity of mitochondrial metabolic pathways based on a colorimetric assay. A comparative metabolic analysis between cell lines or primary cells allows to establish a specific metabolic profile and to detect possible alterations of the mitochondrial function of a tumor cell. Moreover, the direct measurements of electron flux triggered by metabolic pathway activation could highlight targets for potential drug candidates. [less ▲]

The HDAC6 inhibitor 7b induces BCR-ABL ubiquitination and downregulation and synergizes with imatinib to trigger apoptosis in chronic myeloid leukemia

in Pharmacological Research (2020), 160

Despite the discovery of tyrosine kinase inhibitors (TKIs) for the treatment of breakpoint cluster region-Abelson (BCR-ABL)+ cancer types, patients with chronic myeloid leukemia (CML) treated with TKIs ... [more ▼]

Despite the discovery of tyrosine kinase inhibitors (TKIs) for the treatment of breakpoint cluster region-Abelson (BCR-ABL)+ cancer types, patients with chronic myeloid leukemia (CML) treated with TKIs develop resistance and severe adverse effects. Combination treatment, especially with a histone deacetylase (HDAC) 6 inhibitor (HDAC6i), appears to be an attractive option to prevent TKI resistance, considering the potential capacity of an HDAC6i to diminish BCR-ABL expression. We first validated the in vivo anti-cancer potential of the compound 7b by significantly reducing the tumor burden of BALB/c mice xenografted with K-562 cells, without notable organ toxicity. Here, we hypothesize that the HDAC6i compound 7b can lead to BCR-ABL downregulation in CML cells and sensitize them to TKI treatment. The results showed that combination treatment with imatinib and 7b resulted in strong synergistic caspase-dependent apoptotic cell death and drastically reduced the proportion of leukemia stem cells, whereas this treatment only moderately affected healthy cells. Ultimately, the combination significantly decreased colony formation in a semisolid methylcellulose medium and tumor mass in xenografted zebrafish compared to each compound alone. Mechanistically, the combination induced BCR-ABL ubiquitination and downregulation followed by disturbance of key proteins in downstream pathways involved in CML proliferation and survival. Taken together, our results suggest that an HDAC6i potentiates the effect of imatinib and could overcome TKI resistance in CML cells. [less ▲]

Tetrahydrobenzimidazole TMQ0153 triggers apoptosis, autophagy and necroptosis crosstalk in chronic myeloid leukemia

in Cell Death and Disease (2020), 11(2),

By comparing imatinib-sensitive and -resistant chronic myeloid leukemia (CML) cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase ... [more ▼]

By comparing imatinib-sensitive and -resistant chronic myeloid leukemia (CML) cell models, we investigated the molecular mechanisms by which tetrahydrobenzimidazole derivative TMQ0153 triggered caspase-dependent apoptosis at low concentrations accompanied by loss of mitochondrial membrane potential (MMP) and increase of cytosolic free Ca2+ levels. Interestingly, at higher concentrations, TMQ0153 induced necroptotic cell death with accumulation of ROS, both preventable by N-acetyl-L-cysteine (NAC) pretreatment. At necroptosis-inducing concentrations, we observed increased ROS and decreased ATP and GSH levels, concomitant with protective autophagy induction. Inhibitors such as bafilomycin A1 (baf-A1) and siRNA against beclin 1 abrogated autophagy, sensitized CML cells against TMQ0153 and enhanced necroptotic cell death. Importantly, TMQ153-induced necrosis led to cell surface exposure of calreticulin (CRT) and ERp57 as well as the release of extracellular ATP and high mobility group box (HMGB1) demonstrating the capacity of this compound to release immunogenic cell death (ICD) markers. We validated the anti-cancer potential of TMQ0153 by in vivo inhibition of K562 microtumor formation in zebrafish. Taken together, our findings provide evidence that cellular stress and redox modulation by TMQ0153 concentration-dependently leads to different cell death modalities including controlled necrosis in CML cell models. © 2020, The Author(s). [less ▲]

Identification of genes under dynamic post-transcriptional regulation from time-series epigenomic data

in Epigenomics (2019)

Aim: Prediction of genes under dynamic post-transcriptional regulation from epigenomic data. Materials & methods: We used time-series profiles of chromatin immunoprecipitation-seq data of histone ... [more ▼]

Aim: Prediction of genes under dynamic post-transcriptional regulation from epigenomic data. Materials & methods: We used time-series profiles of chromatin immunoprecipitation-seq data of histone modifications from differentiation of mesenchymal progenitor cells toward adipocytes and osteoblasts to predict gene expression levels at five time points in both lineages and estimated the deviation of those predictions from the RNA-seq measured expression levels using linear regression. Results & conclusion: The genes with biggest changes in their estimated stability across the time series are enriched for noncoding RNAs and lineage-specific biological processes. Clustering mRNAs according to their stability dynamics allows identification of post-transcriptionally coregulated mRNAs and their shared regulators through sequence enrichment analysis. We identify miR-204 as an early induced adipogenic microRNA targeting Akr1c14 and Il1rl1. [less ▲]

APPROACHES FOR IDENTIFICATION OF TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL REGULATORS OF MESENCHYMAL STEM CELL DIFFERENTIATION USING TIME-SERIES EPIGENOMIC DATA

Doctoral thesis (2017)

Gene regulatory networks (GRNs) control cellular differentiation and development and recapitulate the physical interactions between transcription factors (TFs) and their influence on their target genes ... [more ▼]

Gene regulatory networks (GRNs) control cellular differentiation and development and recapitulate the physical interactions between transcription factors (TFs) and their influence on their target genes that ultimately results into a defined cell phenotype. In addition, cellular differentiation represents the path a cell undergoes through multiple stages before reaching a terminally differentiated state and is by nature dynamic. Moreover, epigenetic regulation as well as post-transcriptional control of gene expression are critical for faithful cellular phenotype. Cellular differentiation of progenitor cells into their daughter cells provide a dynamic controllable system to study the epigenetic mechanisms as well as the transcriptional output that take place towards cellular specifications, and the TFs and non-coding RNAs that dictate their differentiation. Here, we have generated time-series transcriptomic and epigenomic data during the differentiation of bone marrow stromal cells towards adipocytes and osteoblasts and characterized a novel approach called EPIC-DREM to construct dynamic GRNs of adipocytes and osteoblasts. In order to focus on shared transcriptional regulators of early commitment of bone marrow stromal cells towards adipocytes and osteoblasts, we have concentrated our analysis on dynamic super-enhancers to prioritize the identified TFs and discovered aryl hydrocarbon receptor (AHR) as a transcriptional regulator of the multipotent state. In addition, the generated of time-series epigenomic data were used as input for linear regression analysis that allowed to predict genes that are dynamically controlled by post-transcriptional regulators such as microRNAs (miRs). Indeed, genes that differ from their predicted expression level as assessed by the residuals of the linear regression model can be informative about their mRNA stability. In order to decipher genes that are under dynamic post-transcriptional control, the standard deviation of gene’s residuals was taken as a dynamic measure of changes in mRNA stability and clustering analysis coupled to microRNA motifs enrichment analysis allowed to identify post-transcriptionally co-regulated mRNAs. Based on the linear regressions analysis, miR-204 was identified as a potential regulator of adipogenesis. Integration of these types of data can contribute to the understanding of transcriptional and post-transcriptional control of cell differentiation and the here established approaches for key regulators identification can be widely applied to study other cell states transitions. [less ▲]