Cosmogenic Isotope Production in Argon

<p>This presentation was used for the Low-Radioactivity Underground Argon Workshop held at Pacific Northwest National Laboratory in Richland, Washington on March 19 - 20, 2018.</p

Design, Synthesis, and Osteogenic Activity of Daidzein Analogs on Human Mesenchymal Stem Cells

Osteoporosis is caused by an overstimulation of osteoclast activity and the destruction of the bone extracellular matrix. Without the normal architecture, osteoblast cells are unable to rebuild phenotypically normal bone. Hormone replacement therapy with estrogen has been effective in increasing osteoblast activity but also has resulted in the increased incidence of breast and uterine cancer. In this study we designed and synthesized a series of daidzein analogs to investigate their osteogenic induction potentials. Human bone marrow derived mesenchymal stem cells (MSCs) from three different donors were treated with daidzein analogs and demonstrated enhanced osteogenesis when compared to daidzein treatment. The enhanced osteogenic potential of these daidzein analogs resulted in increased osterix (Sp7), alkaline phosphatase (ALP), osteopontin (OPN), and insulin-like growth factor 1 (IGF-1), which are osteogenic transcription factors that regulate the maturation of osteogenic progenitor cells into mature osteoblast cells

Novel application of the published kinase inhibitor set to identify therapeutic targets and pathways in triple negative breast cancer subtypes

<div><p>Triple negative breast cancers (TNBCs) have high recurrence and metastasis rates. Acquisition of a mesenchymal morphology and phenotype in addition to driving migration is a consequential process that promotes metastasis. Although some kinases are known to regulate a mesenchymal phenotype, the role for a substantial portion of the human kinome remains uncharacterized. Here we evaluated the Published Kinase Inhibitor Set (PKIS) and screened a panel of TNBC cell lines to evaluate the compounds’ effects on a mesenchymal phenotype. Our screen identified 36 hits representative of twelve kinase inhibitor chemotypes based on reversal of the mesenchymal cell morphology, which was then prioritized to twelve compounds based on gene expression and migratory behavior analyses. We selected the most active compound and confirmed mesenchymal reversal on transcript and protein levels with qRT-PCR and Western Blot. Finally, we utilized a kinase array to identify candidate kinases responsible for the EMT reversal. This investigation shows the novel application to identify previously unrecognized kinase pathways and targets in acquisition of a mesenchymal TNBC phenotype that warrant further investigation. Future studies will examine specific roles of the kinases in mechanisms responsible for acquisition of the mesenchymal and/or migratory phenotype.</p></div

Immunofluorescence staining to observe morphological changes after treatment with GSK inhibitors.

<p>(A) Apotome-based microscopic imaging of triple negative MDA-MB-231 cells treated with DMSO (control; panel <i>a</i>), GSK198271A, GSK350559A, GSK494610A, GSK346294A, GSK448459A, GSK237700A, GSK809897X, and GSK1010829B. The samples were pretreated for 72 hours before staining and experiment was performed in triplicate. A phalloidin stain was used to highlight actin filaments (red), DAPI stained for the nucleus (blue). (B) Quantitative morphological analysis of MDA-MB-231 cells after treatment with select GSK compounds. A minimum of 45 cells were quantified per treatment group. Measurements of cellular morphology after immunofluorescence staining and Apotome microscopy imaging are reported. Area is defined as the length of the cell x the width; Circularity = 4(π) x (area/perimeter²). Aspect Ratio = length/width; Nuclear Area Fraction = (Area of Nucleus/Area of Cell). Greater area, cell circularity, aspect ratio and nuclear area fraction are associated with a mesenchymal phenotype due to cytoskeletal rearrangement [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177802#pone.0177802.ref031" target="_blank">31</a>]. For quantification experiments, N ≥ 45, error bars represent SEM and significantly different * p < 0.05, *** p < 0.001.</p

Analysis of structurally similar PLK inhibitors and anti-malarial inhibitors.

<p>(A) Three small molecule PLK inhibitors (GSK346294A, GSK448459A and GSK237700A) are similar in structure and have similar effects on reversal of a mesenchymal phenotype as indicated by an increase in CDH1 and suppression of cellular migration. (B) Two structurally similar anti-malarial inhibitors (GSK198271A, GSK350559A) identified in the initial screen alter cellular morphology, but only GSK198271A increases CDH1 expression. These data indicate there is a potential off-target effect that drives the observed effects of the inhibitors. Pharmacophores are outlined in blue. Compounds selected for further in-depth analyses altered the mesenchymal phenotype by affecting gene expressions or suppression of cellular migration.</p

Common targets and unique targets of GSK198271A and GSK350559A.

<p>Unique targets of GSK198271A include EGFR, HER2, TAOK2, and the SRC family kinases HCK, LYN, LCK, CSK and c-SRC. Data is shown as percent activity, or the remaining activity of the kinase in the presence of the inhibitor.</p

Cellular morphology quantification calculations.

<p>Calculations and measurements are described with the associated formulas based on cellular dimensions of individual cells.</p

Cell morphology screen of small molecule kinase inhibitors from PKIS library induce MET in mesenchymal breast cancer cell lines.

<p>(A) Three phenotypically mesenchymal breast cancer cell lines (MDA-MB-231, MDA-MB-157 and BT549) were treated with small molecule kinase inhibitors provided in the GlaxoSmithKline PKIS library or vehicle (DMSO) for 72 hours’ treatment (1μM). Schematic representation of the broad screen approach. (B) Crystal violet staining techniques were used to observe changes in cellular morphology after treatment. Images were captured with Nikon Camera at 100X magnification. Representative inset images of select hit compounds are shown; additional hit compounds as well as full images are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177802#pone.0177802.s002" target="_blank">S1 Fig</a>. LBH-589 treatment (100 nM) was used as a positive control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177802#pone.0177802.ref027" target="_blank">27</a>]. Morphological changes to a more epithelial phenotype included rounder cells, cells that congregate (indicative of increased cell-cell contacts) and fewer fibroblastic-like cells.</p

The pyrazolopyridazine GSK198271A promotes MET.

<p>(A) Western blot analyses of MDA-MB-231 cells after treatment with GSK198271 confirms CDH1 protein expression in increased. Full length western blots are found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177802#pone.0177802.s004" target="_blank">S3 Fig</a>. Cells were pre-treated for 72 hours at 1μM; data was normalized to Rho. (B) Treatment of PDX tumors ex vivo with GSK198271 resulted in increased the epithelial marker CDH1 mRNA expression and reduced expression of CDH2 and MMP2, mesenchymal markers. Tumor pieces were treated for 72 hours at 1μM. (C) GSK198271A and not GSK350559A suppresses migration in another TNBC cell line, BT549. (D) Neither GSK198271A nor GSK350559A significantly increase CDH1 expression in BT549 cells. (E) GSK198271A and GSK350559A affected cellular proliferation at drug concentrations greater than 1μM. MDA-MB-231 cells were treated for 72 hours, then crystal violet stained and imaged. Absorbance was measured at 570 nm to obtain dose-response curves. Data was normalized to DMSO and performed in triplicate. (F,G) Representative images are enlarged for 1μM concentrations. Images were taken at 200x magnification using the Nikon color microscope. For qRT-PCR experiments, N = 3, error bars represent SEM and significantly different * p < 0.05, *** p < 0.001.</p

Kinase inhibition induces MET through enhanced MET gene expression and suppression of cellular migration.

<p>(A) Fold change in the epithelial marker CDH1 was observed after MDA-MB-231 cells were treated with 1 μM of select small molecule inhibitors after 72 hours’ pretreatment. Data was obtained by qRT-PCR and normalized both to actin and the vehicle (DMSO)-treated control designated as 1. (B) Transwell migration assays of MDA-MB-231 cells revealed GSK346294A, GSK1010829B and GSK1173862A most significantly suppressed TNBC cellular migration. Migrated cell counts were normalized to vehicle. Images of migrated cells were captured with microscopy at 200X. Cells were counted in eight views of the wells, and migration was performed in triplicate for each treatment. (* p < 0.05, *** p < 0.001). (C, D) Mesenchymal transcription factors c-FOS and SNAI1were also analyzed was observed after MDA-MB-231 cells were treated with 1 μM of select small molecule inhibitors after 72 hours’ pretreatment. Data was obtained by qRT-PCR and normalized both to actin and the vehicle (DMSO)-treated control designated as 1. For all experiments, N = 3, error bars represent SEM and significantly different * p < 0.05, *** p < 0.001.</p