Fig 1.
Salinomycin suppresses cell viability.
TNBC cell lines [(A) MDA-MB-231 and (B) 4T1 cells] were treated with salinomycin (0.5–10 μM) or vehicle (DMSO) for the indicated durations. Viable cells were evaluated by MTS assay. Results are expressed as mean ± SEM and were analyzed by two-way ANOVA followed by Bonferroni’s post hoc test (*p<0.05, ** p<0.01 and *** p<0.001, versus DMSO control). The experiment was independently performed three times (n = 3). Sal, salinomycin.
Fig 2.
Salinomycin induces anoikis-sensitivity.
(A) After exposure to salinomycin (0.5–2 μM) or DMSO for 24–48 h, the sub-G1 population was examined by flow cytometry. The percentage of cells in the sub-G1 fraction (red arrow) is depicted in the plot. (B-D) MDA-MB-231 and (E-F) 4T1 cells were grown in plates that were either uncoated or coated with poly-HEMA (2-hydroxyethyl methacrylate, 10 mg/ml) for 24 h and then treated with salinomycin (0.5–2 μM) or DMSO. (B) Effect of salinomycin on the sub-G1 fraction in anchorage-dependent and -independent conditions. The graph represents the percentage of cells in sub-G1 (++ p<0.01 and # p<0.05). (C) Effect of salinomycin on levels of apoptosis-related factors in MDA-MB-231 anchorage-dependent and -independent cells. Actin was used as a loading control. Quantitative graphs of cleaved caspase-3, cleaved caspase-8, and survivin signal intensity are shown (right panel, + p<0.05 and ## p<0.01). (D) Salinomycin (2 μM, 48 h) significantly increased the number of early and late apoptotic cells in anchorage-independent growth. The graph represents the percentage of annexin V-positive cells (bottom panel, + p<0.05). (E) Effect of salinomycin on levels of apoptosis-related factors in 4T1 anchorage-dependent and -independent cells. Quantification of cleaved caspase-3 and survivin are shown (right panel, +++ p<0.001 and ### p<0.001). (F) Early and late apoptotic cells were increased following salinomycin treatment (0.5 μM, 48 h) in 4T1 anchorage-independent growth. The percentage of annexin V-positive cells are shown in the graph (bottom panel, + p<0.05). The results are expressed as mean ± SEM and were analyzed by two-way ANOVA followed by Bonferroni’s post hoc test (+ p<0.05, ++ p<0.01, and +++ p<0.001, versus anchorage-independent DMSO control; # p<0.05, ## p<0.01, and ### p<0.001, versus each concentration; NS, not significant). All experiments were independently performed at least three times (n = 3).
Fig 3.
Salinomycin suppresses STAT3 activation.
(A) Basal levels of STAT3 and phospho-STAT3 (Tyr705) in seven breast cancer cell lines: luminal-type (MCF7 and T47D), HER2-amplified (BT474, MDA-MB-453, and SKBR3), and TNBC (Hs578T and MDA-MB-231). (B) After exposure of MDA-MB-231 cells to salinomycin (1–10 μM) or DMSO for 48 h, STAT3 and phospho-STAT3 protein content were determined by Western blot analysis. A quantitative graph of the phospho-STAT/STAT3 ratio is shown (bottom panel, * p<0.05). (C) Cells were treated with salinomycin (2 μM, 48 h) and immunostained for STAT3 (1:100, green) or phospho-STAT3 (1:100, green), with DAPI nuclear staining (blue). F-actin (1:100, red) was used as a cytosolic marker. Nuclear phospho-STAT3 intensity (y-axis) is represented in arbitrary units as defined by the software. (D) Cells were pretreated with IL-6 (0–10 ng/ml) for 1 h before salinomycin treatment (2 μM, 48 h). STAT3 and phospho-STAT3 protein levels were determined by Western blot analysis. Quantification of the phospho-STAT/STAT3 ratio is shown (bottom panel, *** p<0.001 and ### p<0.001). The results are expressed as mean ± SEM (n = 3, independent experiments) and were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test (* p<0.05, ** p<0.01 and *** p<0.001, versus DMSO control; ### p<0.001, versus each treatment).
Fig 4.
Salinomycin inhibits the expression of STAT3 downstream target molecules and impairs cell migration and invasion.
(A) Effect of salinomycin (2 μM, 48 h) on cyclin D1 mRNA and protein content. Quantitative graphs of cyclin D1 mRNA and protein levels are shown (bottom panel, Student’s t-test, ** p<0.01). (B) Effect of salinomycin on subcellular localization of cyclin D1. Nuclear accumulation of cyclin D1 (1:100, green) was reduced following salinomycin (2 μM, 48 h) treatment. White arrow indicates nuclear cyclin D1; the nuclei were stained with DAPI. (C) Effect of salinomycin on cyclin D1 and survivin protein content in the presence or absence of IL-6 (0–10 ng/ml) in MDA-MB-231 cells. Quantitative graphs of cyclin D1 and survivin protein levels are shown (bottom panel, * p<0.05 and ### p<0.001). (D) MMP-2 and MMP-9 mRNA abundance was analyzed by RT-PCR analysis of total RNA isolated from DMSO- or salinomycin-treated cells (2 μM, 48 h). Quantitative graphs of MMP-2 and MMP-9 mRNA levels are shown (right panel, Student’s t-test, * p<0.05). (E) Effect of salinomycin on cell migration. After salinomycin treatment (0–10 μM), kinetic analysis of cell migration was conducted using an IncuCyte™ Live-Cell Imaging System for the indicated time durations. (a) The kinetic graph of cell migration represents the relative wound density (** p<0.01). (b) Representative images show wound closure by cell migration at 0 and 24 h in the presence or absence of 2 μM salinomycin. The black lines indicate the initial scratch areas (width, 700–800 μm) and the gray regions represent the empty space not covered by cells. (F) Effect of salinomycin on cell invasion. Images of invading cells were captured with an inverted microscope at ×200 magnification. Enlarged images from selected areas are shown. The graph represents the percentage of invaded cells (right panel, Student’s t-test, ** p<0.01). (G-H) Effect of salinomycin (0.5–2 μM, 24 h) on STAT3 and phospho-STAT3 protein levels in (G) MDA-MB-231 and (H) 4T1 cells in anchorage-dependent and -independent growth. Quantitative graph of intensity ratio of STAT3 and phospho-STAT3 is shown (bottom panel). The results are presented as mean ± SEM and were analyzed by two-way ANOVA followed by Bonferroni’s post hoc test (* p<0.05, ** p<0.01, ***p<0.001, versus anchorage-dependent DMSO control; ++ p<0.01 and +++ p<0.001, versus anchorage-independent DMSO control; # p<0.05, ## p<0.01 and ### p<0.001, versus each concentration). All experiments were independently performed at least three times (n = 3).
Fig 5.
Salinomycin decreases the CD44+/CD24- stem-like population during anchorage-independent growth and inhibits mammosphere formation.
(A) The CD44+/CD24- stem-like population in several breast cancer cell lines [luminal-type (MCF7 and T47D), HER2-amplified (BT474, MDA-MB-453, and SKBR3) and TNBC (MDA-MB-231)] was evaluated by flow cytometry. (B) Effect of salinomycin (2 μM, 48 h) on CD44+/CD24- levels in anchorage-dependent and -independent conditions. The graph represents the percentage of CD44+/CD24- cells (right panel). Data are expressed as mean ± SEM (n = 3, independent experiments) and were analyzed by two-way ANOVA followed by Bonferroni’s post hoc test (++ p<0.01, versus anchorage-independent DMSO control; NS, not significant). (C) MDA-MB-231 and 4T1 mammospheres were cultured for 5 days in serum-free suspension conditions in the presence or absence of salinomycin (2 μM). Graphs represents the number (per 104 cells) and volumes (mm3) of mammospheres (bottom panel, Student’s t-test, ** p<0.01).
Fig 6.
Combination effect of salinomycin and STAT3 inhibitors on apoptosis, migration, and CD44+/CD24- stem-like population.
(A) Cells were treated with salinomycin (2 μM, 48 h) and/or S3I-201 (50 μM) or LLL12 (1 μM) and annexin V/PI analysis was performed. Quantitative graph of annexin V-positive population is shown (bottom panel, *** p<0.001 and ### p<0.001). (B) Effect of combined treatment with salinomycin and S3I-201 on protein expression of STAT3, phospho-STAT3, cyclin D1, and apoptosis-related factors. (C) Cells were co-treated with salinomycin (2 μM) and S3I-201 (50 μM) or LLL12 (1 μM) and the relative wound density was assessed over 24 h. Quantitative graph of relative migration is presented (** p<0.01 and ## p<0.01). (D) Effect of salinomycin (2 μM, 48 h) combined with S3I-201 (50 μM) or LLL12 (1 μM) on the CD44+/CD24- stem-like population. The graph represents the percentage of CD44+/CD24- cells (bottom panel, * p<0.05 and ## p<0.01). The results were expressed as mean ± SEM and analyzed by one-way ANOVA followed by Bonferroni’s post hoc test (* p<0.05, ** p<0.01, and *** p<0.001, versus DMSO control; ## p<0.01 and ### p<0.001, versus salinomycin only). All experiments were independently performed at least three times (n = 3).