Figure 1.
Effect of salinomycin on cell viability in colon and breast cancer cell lines.
The cell viability was determined by MTT and colony forming assays. (A) Colon cancer cell lines, 72 hours post treatment (p<0.001, unifactorial ANOVA); (B) breast cancer cell lines, 72 hours post treatment (p<0.001, unifactorial ANOVA). (C) 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). (D) 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).
Figure 2.
Induction of cell death by salinomycin in colon and breast cancer cell lines.
(A) DNA fragmentation is induced after 48 hours of salinomycin treatment. DNA fragmentation was measured by flow cytometry as a sub-G1 cell fraction after propidium iodide staining (*: p<0.05; **: p<0.01; ***: p<0.001 by t-test). (B) Measurement of the mitochondrial potential using JC-1 staining, determined as the ratio of red (FL2, JC1-aggregates) and green fluorescence (FL1, monomeric form) (*: p<0.05; **: p<0.01; ***: p<0.001 by t-test). (C) Staining of colon cancer cell line SW480 with JC-1 visualization via fluorescence microscopy. Cells without salinomycin treatment show orange fluorescence, indicating an intact mitochondrial potential. Apoptotic cells with decreased mitochondrial potential show green fluorescence.
Figure 3.
Impact of salinomycin on caspase activation.
Caspase activities were measured after treatment of breast and colon cancer cell lines with salinomycin for 28 hours. (A) Caspase 9 activity measured with Z-LEHD-aminoluciferin as a substrate; (B) Caspase 8 activity measured with Z-LETD-aminoluciferin as a substrate; (C) 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. (D) 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. (E) 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. (F) 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.
Figure 4.
Induction of autophagy by salinomycin.
(A) Electron microscopic images of MCF-7 cells 48 hours after treatment with 10 µM salinomycin or solvent control. Note the vacuoles formed in the cytoplasm (arrow). (B) Salinomycin induced uptake of monodansylcadaverine (MDC) in SW620, RKO and MCF-7 cells 8 hours after salinomycin treatment, in comparison to doxorubicin. Cells with a punctate staining pattern were counted. (*: p<0.05; **: p<0.01, ***: p<0.001 by t-test, compared to the solvent control). (C) Confocal microscopy of cells transfected with GFP-LC3 16 hours after treatment as indicated (sali: 2.5 µM salinomycin). Upper row: MCF-7, bottom row: SW620. (D) Induction of the autophagy-related genes Beclin-1, ATG7, and ATG12, and processing of LC3B after salinomycin treatment of MCF-7. Cells were treated for the indicated durations with 2.5 µM or 10 µM salinomycin, and analyzed for autophagy-specific markers by Western blot. β-actin was used as a loading control. (E) Expression of the autophagy-related gene Beclin-1, and processing of LC3B after salinomycin treatment in SW620. Cells were treated for the indicated durations with 2.5 µM salinomycin, and analyzed for autophagy-specific markers by Western blot. β-Actin was used as a loading control. (F) Processing of LC3 in SW480 after salinomycin treatment in the presence or absence of 0.25 mg/ml catalase and 20 µM SP600125. 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.
Figure 5.
Partial protection against salinomycin toxicity through inhibition of autophagy.
Cell death was measured in SW620 by flow cytometry using the ViaCount reagent 24 hours after treatment with 10 µM salinomycin, or solvent control. (A) Cells were transfected with siRNA against ATG7, or control oligos, and treated with salinomycin 72 hours after transfection. (B) Protection against cell death induction by salinomycin after 3 hours pre-treatment with 1 µM Wortmannin.
Figure 6.
Induction of reactive oxygen species by salinomycin treatment.
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. (A) H2O2 was detected through oxidation of dihydrorhodamine and (B) O2• through oxidation of dihydroethidium. (*: p<0.05; **: p<0.01, ***: p<0.001 by t-test). (C) 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. (D) 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.
Figure 7.
Activation of the JNK pathway by salinomycin.
(A) Exposure to 2.5 µM salinomycin induced the expression and activation of JNK, as well as its target JUN in MCF-7 cells. (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. (C) 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. (D) 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).
Figure 8.
Decreased autophagy induction through salinomycin by inhibition of JNK.
(A) 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) 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.