The %age of GS-expressing tumors with nuclear (N), cytosolic (C) and normal membranous (M) β-catenin expression is indicated

Numbers on top indicate the total amount of HCCs in each group.<p><b>Copyright information:</b></p><p>Taken from "Correlation between β-catenin mutations and expression of Wnt-signaling target genes in hepatocellular carcinoma"</p><p>http://www.molecular-cancer.com/content/7/1/21</p><p>Molecular Cancer 2008;7():21-21.</p><p>Published online 18 Feb 2008</p><p>PMCID:PMC2287186.</p><p></p

Salinomycin Induces Autophagy in Colon and Breast Cancer Cells with Concomitant Generation of Reactive Oxygen Species

<div><h3>Background</h3><p>Salinomycin is a polyether ionophore antibiotic that has recently been shown to induce cell death in human cancer cells displaying multiple mechanisms of drug resistance. The underlying mechanisms leading to cell death after salinomycin treatment have not been well characterized. We therefore investigated the role of salinomycin in caspase dependent and independent cell death in colon cancer (SW480, SW620, RKO) and breast cancer cell lines (MCF-7, T47D, MDA-MB-453).</p> <h3>Methodology/Principal Findings</h3><p>We detected features of apoptosis in all cell lines tested, but the executor caspases 3 and 7 were only strongly activated in RKO and MDA-MB-453 cells. MCF-7 and SW620 cells instead presented features of autophagy such as cytoplasmic vacuolization and LC3 processing. Caspase proficient cell lines activated autophagy at lower salinomycin concentrations and before the onset of caspase activation. Salinomycin also led to the formation of reactive oxygen species (ROS) eliciting JNK activation and induction of the transcription factor <em>JUN</em>. Salinomycin mediated cell death could be partially inhibited by the free radical scavenger N-acetyl-cysteine, implicating ROS formation in the mechanism of salinomycin toxicity.</p> <h3>Conclusions</h3><p>Our data indicate that, in addition to its previously reported induction of caspase dependent apoptosis, the initiation of autophagy is an important and early effect of salinomycin in tumor cells.</p> </div

Activation of the JNK pathway by salinomycin.

<p>(<b>A</b>) Exposure to 2.5 µM salinomycin induced the expression and activation of JNK, as well as its target JUN in MCF-7 cells. (<b>B</b>) Increased JNK kinase activity after salinomycin treatment in MCF-7. The JNK kinase assay was carried out after treatment with 2.5 µM salinomycin for the indicated durations; recombinant JUN was used as the substrate. (<b>C</b>) Densitometric analysis of the kinase assay. The difference to the untreated control is significant for the 16 hour time point (*: p<0.05). Error bars: standard deviation of three experiments. (<b>D</b>) Measurement by ELISA of JNK-phosphorylation after treatment of MCF-7 with 2.5 µM salinomycin, compared to medium control. Cells were pre-treated with 0.25 mg/ml catalase or medium control for 3 hours. Combined results from 2 independent experiments are shown (*: p<0.05).</p

Partial protection against salinomycin toxicity through inhibition of autophagy.

<p>Cell death was measured in SW620 by flow cytometry using the ViaCount reagent 24 hours after treatment with 10 µM salinomycin, or solvent control. (<b>A</b>) Cells were transfected with siRNA against ATG7, or control oligos, and treated with salinomycin 72 hours after transfection. (<b>B</b>) Protection against cell death induction by salinomycin after 3 hours pre-treatment with 1 µM Wortmannin.</p

Impact of salinomycin on caspase activation.

<p>Caspase activities were measured after treatment of breast and colon cancer cell lines with salinomycin for 28 hours. (<b>A</b>) Caspase 9 activity measured with Z-LEHD-aminoluciferin as a substrate; (<b>B</b>) Caspase 8 activity measured with Z-LETD-aminoluciferin as a substrate; (<b>C</b>) Caspase 3/7 activity measured in colon cancer cell lines with Z-DEVD-aminoluciferin as a substrate. Measurements were in MCF-7 cells were omitted as these cells are caspase 3-negative. Significant increases in measured luminescence in comparison to the untreated cells are indicated in the graphs (*: p<0.05; **: p<0.01, ***: p<0.001 by t-test). RKO cells were most sensitive to salinomycin treatment for all the measured caspases. (<b>D</b>) Western blot analysis of caspase status. Procaspase 3 and procaspase 9 expression were determined in SW480, SW620, RKO, and MCF-7 cells. β-Actin was used as a loading control. (<b>E</b>) Caspase 3 status in T47D and MDA-MB-453 cells. Procaspase 3 expression was determined by Western blot; α-Tubulin was used as a loading control. (<b>F</b>) Caspase 3/7 activity measured in breast cancer cell lines with Z-DEVD-aminoluciferin as a substrate. Caspase 3 activity was not measured in MCF-7 as these cells are caspase 3-negative.</p

Effect of salinomycin on cell viability in colon and breast cancer cell lines.

<p>The cell viability was determined by MTT and colony forming assays. (<b>A</b>) Colon cancer cell lines, 72 hours post treatment (p<0.001, unifactorial ANOVA); (<b>B</b>) breast cancer cell lines, 72 hours post treatment (p<0.001, unifactorial ANOVA). (<b>C</b>) Cell viability assay performed 6 days post treatment. Colon and breast cancer cell lines were treated with the indicated concentrations of salinomycin. Salinomycin reduced the cell viability in a time and concentration dependent manner (p<0.001, unifactorial ANOVA). (<b>D</b>) Sensitivity to salinomycin in the colony forming assay. Assays were performed in triplicates. After incubation for 10 days, the plates were stained with crystal violet. The reduction of colony number depended on the salinomycin concentration and the cell line (*: p<0.05; **: p<0.01 by unifactorial anova).</p

Induction of reactive oxygen species by salinomycin treatment.

<p>Production of ROS was measured by flow cytometry in RKO, MCF-7 and SW620 cells 30 hours after treatment with the indicated concentrations of salinomycin or solvent control. Treatment with 1 mM H₂O₂ for 1 hour served as a positive control. (<b>A</b>) H₂O₂ was detected through oxidation of dihydrorhodamine and (<b>B</b>) O₂• through oxidation of dihydroethidium. (*: p<0.05; **: p<0.01, ***: p<0.001 by t-test). (<b>C</b>) Effect of catalase on salinomycin toxicity by MTT assay. Cells were pre-treated for 3 hours with 0.25 mg/ml catalase, followed by the addition of the indicated concentration of salinomycin (1 µM; 2.5 µM) or solvent control (ctrl). Absorbance was measured 72 hours after the addition of salinomycin. (<b>D</b>) Processing of LC3 in MCF-7 and SW620 after salinomycin treatment in the presence or absence of 0.25 mg/ml catalase. Cells were treated with 2.5 µM salinomycin for the indicated duration; the relative level of LC3-II in comparison to tubulin is indicated below the figure.</p

Decreased autophagy induction through salinomycin by inhibition of JNK.

<p>(<b>A</b>) Confocal microscopy of cells transfected with GFP-LC3 16 hours after treatment as indicated (sali: 2.5 µM salinomycin; SP: 20 µM SP600125). Upper row: MCF-7, bottom row: SW620. (<b>B)</b> Effect of pre-treatment with 20 µM SP600125 on LC3 processing after addition of 2.5 µM salinomycin for the indicated durations in SW620 cells. The relative level of LC3-II in comparison to tubulin is indicated below the figure.</p

Expression of ANOs in the human TG and DRG.

<p>Transcription of ANO channel members was detected in the TG and DRG.</p

The OR6B2 protein is localized to satellite cells in human DRG.

<p>Immunohistochemical staining of human DRG sections with the OR6B2 antibody indicated OR6B2 protein expression in the neuron-surrounding satellite cells. DAPI staining (blue) was used to determine the number and localization of cell nuclei. The secondary antibody alone did not produce any staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128951#pone.0128951.s004" target="_blank">S4 Fig</a>). Scale bars: 10 μm.</p