41 research outputs found
Anion-Exchange Chromatography Coupled to High-Resolution Mass Spectrometry: A Powerful Tool for Merging Targeted and Non-targeted Metabolomics
In
this work, simultaneous targeted metabolic profiling by isotope
dilution and non-targeted fingerprinting is proposed for cancer cell
studies. The novel streamlined metabolomics workflow was established
using anion-exchange chromatography (IC) coupled to high-resolution
mass spectrometry (MS). The separation time of strong anion-exchange
(2 mm column, flow rate 380 μL min<sup>–1</sup>, injection
volume 5 μL) could be decreased to 25 min for a target list
comprising organic acids, sugars, sugar phosphates, and nucleotides.
Internal standardization by fully <sup>13</sup>C labeled <i>Pichia
pastoris</i> extracts enabled absolute quantification of the
primary metabolites in adherent cancer cell models. Limits of detection
(LODs) in the low nanomolar range and excellent intermediate precisions
of the isotopologue ratios (on average <5%, <i>N</i> = 5, over 40 h) were observed. As a result of internal standardization,
linear dynamic ranges over 4 orders of magnitude (5 nM–50 μM, <i>R</i><sup>2</sup> > 0.99) were obtained. Experiments on drug-sensitive
versus resistant SW480 cancer cells showed the feasibility of merging
analytical tasks into one analytical run. Comparing fingerprinting
with and without internal standard proved that the presence of the <sup>13</sup>C labeled yeast extract required for absolute quantification
was not detrimental to non-targeted data evaluation. Several interesting
metabolites were discovered by accurate mass and comparing MS2 spectra
(acquired in ddMS2 mode) with spectral libraries. Significant differences
revealed distinct metabolic phenotypes of drug-sensitive and resistant
SW480 cells
XB-induced cell-cycle blockade.
<p>Semi-confluent cultures of SW480 (<b>A</b>) and Caco2 (<b>B</b>) cells were exposed to the indicated concentrations of XB. 48 hours later nuclei were isolated for the analysis of cell cycle distribution by FACS analysis. The results shown are the mean±SD pooled from three independent experiments. Protein lysates were harvested 20 hours after exposure of SW480 (<b>C</b>) and Caco2 (<b>D</b>) cells to 20 µM XB and levels of Cyclins A, B1, and E as well as of FoxM1 and phospho-Histone H3 were analysed by western blotting. The figure shows representative examples of two independent experiments.</p
XB-induced active cell death.
<p><b>A, B, C, D</b>: In a parallel experiment cultures were fixed after 48 hours of XB-exposure and stained with Hoechst 33258 for visualisation of nuclear morphology (<b>A</b>: control SW480, <b>B</b>: XB treated SW480, <b>C</b>: control CaCo2, <b>D</b>: XB treated CaCo2). <b>E</b>: Semi-confluent cultures of SW480 were exposed to the indicated concentrations of XB and harvested 48 and 72 hours later for determination of mitochondrial membrane potential (MMP). The results shown are the mean±SD pooled from three independent experiments.</p
XB-induced cell loss.
<p>Semiconfluent cell cultures of HCT116, DLD1, CaCo2, SW620, SW480 (<b>A</b>) and F331 and LT97 (<b>B</b>) were exposed to increasing concentrations of XB diluted into serum-free treatment medium. Viability was determined 48 hours later by MMT assay. The results shown are the mean±SD pooled from three independent experiments performed in triplicates. *, ** and *** indicate a significant difference as compared to control at p≤0.05, 0.01 and 0.001, respectively.</p
Sensitivity of CRC cell lines to XB.
*<p>Very wide indicates that the growth response curve declined too gradual for the graph pad prism software to calculate a 95% CI.</p
XB does not induce classical apoptosis.
<p>Protein lysates were harvested 20 hours after exposure to 20 µM XB and levels of Bcl<sub>xl</sub>, Bcl<sub>2</sub>, activated Caspase 3, PARP and cleaved PARP (<b>A</b>) as well as pro Caspase 2 and activated Caspase 2 (<b>B</b>) were analysed by western blotting. The figure shows representative examples of two independent experiments.</p
Additional file 3: Figure S3. of Distinct activity of the bone-targeted gallium compound KP46 against osteosarcoma cells - synergism with autophagy inhibition
KP46 treatment synergizes with the autophagy inhibitor chloroquine. Cell viability after combined treatment of OS cells with KP46 and chloroquine for 72 h at the indicated concentrations was determined by MTT survival assay. CI values for HOS and SAOS-2 cells derived from two independent experiments in triplicate are shown representatively. The respective growth curves are shown in Fig. 5d. (PDF 160 kb
Functional Classification of Cellular Proteome Profiles Support the Identification of Drug Resistance Signatures in Melanoma Cells
Drug
resistance is a major obstacle in melanoma treatment. Recognition
of specific resistance patterns, the understanding of the patho-physiology
of drug resistance, and identification of remaining options for individual
melanoma treatment would greatly improve therapeutic success. We performed
mass spectrometry-based proteome profiling of A375 melanoma cells
and HeLa cells characterized as sensitive to cisplatin in comparison
to cisplatin resistant M24met and TMFI melanoma cells. Cells were
fractionated into cytoplasm, nuclei and secretome and the proteome
profiles classified according to Gene Ontology. The cisplatin resistant
cells displayed increased expression of lysosomal as well as Ca<sup>2+</sup> ion binding and cell adherence proteins. These findings
were confirmed using Lysotracker Red staining and cell adhesion assays
with a panel of extracellular matrix proteins. To discriminate specific
survival proteins, we selected constitutively expressed proteins of
resistant M24met cells which were found expressed upon challenging
the sensitive A375 cells. Using the CPL/MUW proteome database, the
selected lysosomal, cell adherence and survival proteins apparently
specifying resistant cells were narrowed down to 47 proteins representing
a potential resistance signature. These were tested against our proteomics
database comprising more than 200 different cell types/cell states
for its predictive power. We provide evidence that this signature
enables the automated assignment of resistance features as readout
from proteome profiles of any human cell type. Proteome profiling
and bioinformatic processing may thus support the understanding of
drug resistance mechanism, eventually guiding patient tailored therapy
Additional file 2: Figure S2. of Distinct activity of the bone-targeted gallium compound KP46 against osteosarcoma cells - synergism with autophagy inhibition
Induction of large acidic vesicles by KP46 in OS cells and impact of obatoclax. (A) HOS cells were treated with KP46 alone or in combination with obatoclax as indicated. Photomicrographs were taken after 48 h drug exposure in phase contrast setting. Size bar, 10 μm. (B) HOS cells treated with KP46 for 24 h at the indicated concentrations in combination with obatoclax and stained with AO. Fluorescence images were taken with FITC (green) and TRITC (red) filter sets. Size bar, 10 μm. For the respective KP46 single agent photomicrographs compare Fig. 5a. (PDF 353 kb
Structure-Related Mode-of-Action Differences of Anticancer Organoruthenium Complexes with β‑Diketonates
A series
of organorutheniumÂ(II) chlorido complexes with fluorinated O,O-ligands
[(η<sup>6</sup>-<i>p</i>-cymene)ÂRuÂ(F<sub>3</sub>C-acac-Ar)ÂCl]
(<b>1a</b>–<b>6a</b>) and their respective 1,3,5-triaza-7-phosphaadamantane
(pta) derivatives [(η<sup>6</sup>-<i>p</i>-cymene)ÂRuÂ(F<sub>3</sub>C-acac-Ar)Âpta]ÂPF<sub>6</sub> (<b>1b</b>–<b>6b</b>) were synthesized and fully characterized in both solution
and solid state. All complexes were inactive against nonmalignant
keratinocytes but displayed variable activity against cancer cell
models (ovarian, osteosarcoma). Compounds with a ligand containing
the 4-chlorophenyl substituent (<b>6a</b> and <b>6b</b>) exhibited the strongest anticancer effects. Despite a marginally
lower cellular Ru accumulation compared to the chlorido complexes,
pta analogues showed higher activity especially in the osteosarcoma
model. Reduction of glutathione levels by buthionine sulfoximine (BSO)
significantly enhanced the activity of all compounds with the most
pronounced effects being observed for the pta series resulting in
IC<sub>50</sub> values down to the nanomolar range. While all chlorido
complexes potently induce reactive oxygen species, DNA damage, and
apoptosis, the respective pta compounds widely lacked ROS production
but blocked cell cycle progression in G0/G1 phase