8 research outputs found
The combination of RAD001 and BEZ235 can induce cell death in SK-BR-3 cells.
<p>SK-BR-3 cells were treated with RAD001 and/or BEZ235 at the indicated concentrations and electric impedance was measured every 15 minutes with the xCELLigence system (A). Cell index values were calculated and plotted as a function of time. The time of compound addition is indicated with an arrow, and the continuous vertical line indicates the normalization point. Each data point represents the average of 3 wells ±1 SD. SK-BR-3 cells were treated for 48 h with the indicated doses of RAD001 and/or BEZ235, or for 3 h with Staurosporin. Cells were lysed and endogenous proteins visualized by western blot analysis (B). SK-BR-3 cells were treated for 24 h with the indicated doses of RAD001 and/or BEZ235. Gene expression changes were quantified by microarray analysis, computed as log2 relative ratios (treatment/vehicle control), and subjected to hierarchical clustering along the gene probe axis (C).</p
RAD001 and BEZ235 synergistically inhibit growth of 786-O cells <i>in vitro</i> and <i>in vivo</i>.
<p>786-O cells (PTEN and VHL-deficient renal cell carcinoma) were treated for 72 h with different doses of RAD001 and/or BEZ235, inhibition of cell viability was measured using CellTiter-Glo (A), and Chalice software was used to calculate excess inhibition over Loewe additivity for each RAD001 and/or BEZ235 dose combination (B). Data values represent the mean from 3 wells. Mice bearing 786-O xenografts were dosed daily for 3 weeks with RAD001, BEZ235, or the combination of the two compounds. Mean tumor volumes were measured and are plotted as a function of time (C).</p
Crystallographic data and refinement information.
a<p>Numbers in parenthesis are for the highest resolution shell (3.06-2.90).</p>b<p>R<sub>sym</sub> = Σ|I<sub>h</sub>−h>|/ΣI<sub>h</sub> over all h, where I<sub>h</sub> is the intensity of reflection h.</p>c<p>R<sub>cryst</sub> and R<sub>free</sub> = Σ∥F<sub>o</sub>|−|F<sub>c</sub>∥/Σ|F<sub>o</sub>|, where F<sub>o</sub> and F<sub>c</sub> are observed and calculated amplitudes, respectively. Rfree was calculated using 5% of data excluded from the refinement.</p
Key residues involved in resistance to argyrin B are conserved in EF-G homologues.
<p>The amino acid sequence of <i>P. aeruginosa</i> EF-G1 was used to identify EF-G homologues in the indicated organisms by a BLAST search. EF-G sequences were then aligned using ClustalW, and residues conferring resistance to argyrin B are shown in bold.</p
Co-crystal structure of argyrin B bound to <i>P.aeruginosa</i> EF-G1.
<p>(<b>A</b>) The argyrin B binding pocket localizes to the flexible interface between domains III and V, distinct from the GTP/fusidic acid binding domain (**). (<b>B</b>) Inset view. (<b>C</b>) 2D protein-ligand interaction plot showing the chemical structure of the argyrin B macrocyclic polypeptide and the hydrophobic (cyan) and hydrophilic (yellow) amino-acid residues in EF-G1 which are in binding contact. (<b>D</b>) Interactions between <i>P. aeruginosa</i> EF-G (domain III in yellow and domain V in cyan) and argyrin B (gray). (<b>E</b>) Superposition of Thermus thermophilus EF-G in complex with GTP (magenta), Thermus thermophilus EF-G in complex with the ribosome (ribosome not shown) and fuscidic acid (cyan), and structure of the argyrin B-bound Pseudomonas aeroginosa EF-G (FusA1) (yellow). Superposition was done using domains I and II of each of the protein structures.</p
Susceptibility of representative bacteria and resistant mutants to argyrin B.
<p>Susceptibility determinations were conducted using the broth microdilution protocol as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042657#pone.0042657-Caughlan1" target="_blank">[26]</a>.</p>*<p>Argyrin B was not uniformly soluble and occasionally a small amount of precipitate was visible at concentrations greater than 16–32 µg/ml; therefore values here are reported as susceptibility rather than MIC. Selected on 128<sup>a</sup>, 2<sup>b</sup>, 4<sup>c</sup>, or 16<sup>d</sup> µg/ml argyrin B in solid Mueller-Hinton agar.</p
The mode of action of argyrin B is conserved in mammalian cells.
<p>(<b>A</b>) Cytotoxicity profile of argyrin B across 512 mammalian cell lines showing reduced cell viability with an IC<sub>50</sub> below 1 µM in 18 cell lines (red). (<b>B</b>) Susceptibility to argyrin B (IC<sub>50</sub> and A<sub>max</sub> values) was compared to different cytotoxic agents across the cell line panel by calculating Pearson correlation values. (<b>C</b>) RKO and HCT116 cells were treated for 4 days with 1 µM argyrin B, and total proteins were extracted and analyzed by immunoblotting for SDHA and COX2. (<b>D</b>) Cells were transfected with non-targeting (NT) or GFM1 (encoding mEF-G1) siRNA for 7 days, and total proteins were extracted and analyzed by immunoblotting for mEF-G1 and GAPDH. (<b>E</b>) siRNA-transfected cells were treated for 7 days with increasing doses of Argyrin B or MG132, and cell viability was assessed using CellTiter Glo. A representative example of three independent experiments is shown.</p
Rescue of argyrin B-sensitivity by expression of mEF-G1 L693Q.
<p>(<b>A</b>) mEF-G1 wild-type (WT), S452L, S494F or L693Q were stably over-expressed in HCT116 and RKO, cells were treated for 7 days with increasing doses of argyrin B or MG132, and cell viability was assessed using CellTiter Glo. A representative example of two independent experiments is shown. (<b>B</b>) Increase in IC<sub>50</sub> relative to the parental cell line. Average fold increase was calculated from two independent experiments. (<b>C</b>) HCT116 and RKO stably expressing mEF-G1 WT, S452L, S494F or L693Q were lysed, and total proteins extracted and analyzed by immunoblotting for mEF-G1 and GAPDH. (<b>D</b>) Binding of argyrin B to recombinant human mEF-G1 WT, S452L, S494F or L693Q was measured by Biacore and is depicted relative to recombinant bacterial EF-G.</p