Impact of Mistletoe Triterpene Acids on the Uptake of Mistletoe Lectin by Cultured Tumor Cells

Complementary treatment possibilities for the therapy of cancer are increasing in demand due to the severe side effects of the standard cytostatics used in the first-line therapy. A common approach as a complementary treatment is the use of aqueous extracts of Viscum album L. (Santalaceace). The therapeutic activity of these extracts is attributed to Mistletoe lectins which are Ribosome-inactivating proteins type II. Besides these main constituents the extract of Viscum album L. comprises also a mixture of lipophilic ingredients like triterpene acids of the oleanane, lupane and ursane type. However, these constituents are not contained in commercially available aqueous extracts due to their high lipophilicity and insolubility in aqueous extraction media. To understand the impact of the extract ingredients in cancer therapy, the intracellular uptake of the mistletoe lectin I (ML) by cultured tumor cells was investigated in relation to the mistletoe triterpene acids, mainly oleanolic acid. Firstly, these hydrophobic triterpene acids were solubilized using cyclodextrins ("TT" extract). Afterwards, the uptake of either single compounds (isolated ML and the aqueous "viscum" extract) or in combination with the TT extract (ML+TT, viscumTT), was analyzed. The uptake of ML was studied inTHP-1-, HL-60-, 143B- and Ewing TC-71-cells and determined after 30, 60 and 120 minutes by an enzyme linked immunosorbent assay which quantifies the A-chain of the hololectin. It could be shown that the intracellular uptake after 120 minutes amounted to 20 % in all cell lines after incubation with viscumTT. The studies further revealed that the uptake in THP-1-, HL-60- and Ewing TC-71-cells was independent of the addition of TT extract. Interestingly, the uptake of ML by 143B-cells could only be measured after addition of triterpenes pointing to resistance to mistletoe lectin

Aβ profiles generated by Alzheimer's disease causing PSEN1 variants determine the pathogenicity of the mutation and predict age at disease onset.

Familial Alzheimer’s disease (FAD), caused by mutations in Presenilin (PSEN1/2) and Amyloid Precursor Protein (APP) genes, is associated with an early age at onset (AAO) of symptoms. AAO is relatively consistent within families and between carriers of the same mutations, but differs markedly between individuals carrying different mutations. Gaining a mechanistic understanding of why certain mutations manifest several decades earlier than others is extremely important in elucidating the foundations of pathogenesis and AAO. Pathogenic mutations affect the protease (PSEN/γ-secretase) and the substrate (APP) that generate amyloid β (Aβ) peptides. Altered Aβ metabolism has long been associated with AD pathogenesis, with absolute or relative increases in Aβ42 levels most commonly implicated in the disease development. However, analyses addressing the relationships between these Aβ42 increments and AAO are inconsistent. Here, we investigated this central aspect of AD pathophysiology via comprehensive analysis of 25 FAD-linked Aβ profiles. Hypothesis- and data-driven approaches demonstrate linear correlations between mutation-driven alterations in Aβ profiles and AAO. In addition, our studies show that the Aβ (37 + 38 + 40) / (42 + 43) ratio offers predictive value in the assessment of ‘unclear’ PSEN1 variants. Of note, the analysis of PSEN1 variants presenting additionally with spastic paraparesis, indicates that a different mechanism underlies the aetiology of this distinct clinical phenotype. This study thus delivers valuable assays for fundamental, clinical and genetic research as well as supports therapeutic interventions aimed at shifting Aβ profiles towards shorter Aβ peptides

M2ara: unraveling metabolomic drug responses in whole-cell MALDI mass spectrometry bioassays

Fast computational evaluation and classification of concentration responses for hundreds of metabolites represented by their mass-to-charge (m/z) ratios is indispensable for unraveling complex metabolomic drug actions in label-free, whole-cell Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI MS) bioassays. In particular, the identification of novel pharmacodynamic biomarkers to determine target engagement, potency and potential polypharmacology of drug-like compounds in high-throughput applications requires robust data interpretation pipelines. Given the large number of mass features in cell-based MALDI MS bioassays, reliable identification of true biological response patterns and their differentiation from potentially present measurement artefacts is critical. To facilitate the exploration of metabolomic responses in complex MALDI MS datasets, we present a novel software tool, M2ara. Implemented as a user-friendly R-based shiny application, it enables rapid evaluation of Molecular High Content Screening (MHCS) assay data. Furthermore, we introduce the concept of Curve Response Score (CRS) and CRS fingerprints to enable rapid visual inspection and ranking of mass features. In addition, these CRS fingerprints allow direct comparison of cellular effects among different compounds. Beyond cellular assays, our computational framework can also be applied to MALDI MS-based (cell-free) biochemical assays in general

Used isolated ML and isolated ML + OA concentrations.

<p>Used isolated ML and isolated ML + OA concentrations.</p

Summary of the viscum and TT extract batches.

<p>Summary of the viscum and TT extract batches.</p

Cell viability of HL-60-cells.

<p>The cells were treated with different ML concentrations (see Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153825#pone.0153825.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153825#pone.0153825.t003" target="_blank">3</a>) for 30, 60 and 120 minutes. The isolated ML, three viscum extract batches and the viscum extract batch 161 in combination with TT 161 extract batch (25 μg/mL and 35 μg/mL OA) were used. The viability was determined with Annexin V-APC and propidium iodide by flow cytometry. The values are expressed as percentages of the untreated control cells. Error bars represent the standard deviation of n = 2 experiments.</p

Comparison between 2-HP-β-CD and TT extract.

<p>Comparison between 2-HP-β-CD and TT extract.</p

Comparison between TT extract solubilized with 2-HP-β-CD and solved in MeOH.

<p>Comparison between TT extract solubilized with 2-HP-β-CD and solved in MeOH.</p

ML uptake by 143B cells.

<p>The averages of the uptake were calculated using isolated ML and viscum 155 extract batch in two different ML concentrations alone and in combination with TT 155 extract batch (20 μg/mL OA) (see Tables <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153825#pone.0153825.t002" target="_blank">2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153825#pone.0153825.t003" target="_blank">3</a>). The results are expressed as percentage of the respectively used concentration ± standard deviation of n = 5 experiments. * Significant difference to the isolated ML, α ≤ 0.05. ** Significant difference to the viscum 155 extract, α ≤ 0.05.</p

Intracellular uptake of ML after incubation with viscumTT extract by different cell lines.

<p>Intracellular uptake of ML after incubation with viscumTT extract by different cell lines.</p