To systematically identify FDA approved drugs that exhibit similar activity as salinomycin, a fast computational search of the DrugBank database 4.0 of approved drugs was performed (8). Using the principles of similarity searching with two-dimensional fingerprints, the chemical structure of salinomycin was used as a query to compute the similarity of each of 1,623 compounds in DrugBank using the Molecular ACCess System keys (9) and the Tanimoto coefficient (10), as implemented in Molecular Operating Environment (version 2010.10) (11). The selection of hit compounds was based on similarity values and visual inspection.

The breast cancer cell line MDA-MB-231 was obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium supplemented with 10% of fetal bovine serum (FBS) (both from Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified 5% CO₂ atmosphere. Ivermectin and paclitaxel were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany).

Cells were seeded (2×10³ cells/well) into 96-well microplates (Corning Incorporated, Corning, NY, USA) into 0.1 ml of medium at 37°C for 24 h, and then treated with either ivermectin (0.2, 0.4, 0.8, 1, 5 or 8 µM) or paclitaxel (0.001, 0.1, 10 or 1,000 nM) for 72 h at 37°C. The medium was replaced with fresh medium containing drug or vehicle daily. Equal volumes of ethanol and DMSO vehicles were used for ivermectin and paclitaxel, respectively. Cell viability was evaluated by the colorimetric assay CellTiter 96 AQueous One Solution Cell Proliferation assay (Promega Corporation, Madison, WI, USA) according to the manufacturer's protocol. Following 72 h treatment, the AQueous One Solution reagent was added and incubated for an additional 4 h at 37°C. The optical density was measured at 490 nm, using a plate reader and the effect of each treatment was expressed as a percentage of cell viability relative to the untreated control cells. Mean values and standard deviations were calculated; experiments were performed in triplicate.

MDA-MB-231 cells (4.5×10⁵) were plated in a 25 cm² cell culture flask and incubated for 48 h to allow the cells to proliferate to reach a cell density of 70%. Cells were treated with different doses of ivermectin (0.2, 0.4, 0.8, 1, 5 and 8 µM) for 120 h. Control cells were treated with the corresponding amounts of ethanol (the ivermectin vehicle). After 120 h, cells were collected and cultured in drug-free medium in a dish (2 mm grid; 60×15 mm; Corning Incorporated) for 21 days. Subsequently, colonies were fixed in methanol and acetic acid (3:1 v/v), and stained with 0.4% crystal violet (Sigma-Aldrich; Merck KGaA). The number of colonies on the culture dish were counted with a stereo microscope and quantified using ImageJ software version 1.50f (National Institutes of Health, Bethesda, MD, USA).

MDA-MB-231 cells (4.5×10⁵) were grown in a 25 cm² cell culture flask and incubated in DMEM/F12 medium, 10% fetal bovine serum, penicillin (100 U/ml) and streptomycin (1.0 mg/ml) at 37°C in humidified atmosphere of 5% CO₂ for 48 h, to allow the cells to proliferate to a confluence of ~80%. Cells were collected and sub-cultured (1×10³ cells) in 24-well ultra-low attachment multiwell plates (Corning Costar; Corning Incorporated) for 15 days with 1 ml MammoCult Basal Medium plus supplements (STEMCELL Technologies, Inc., Vancouver, BC, Canada), 0.48 µg/ml hydrocortisone and 4 µg/ml heparin, at 37°C in a humidified atmosphere of 5% CO₂ and 95% air. The medium was changed every 2 days. Following 15 days incubation, the spheroids (confluence, ~60–70%) were dispersed and stained with 0.4% trypan blue to assess the cell viability and counted with a TC10 Automated Cell Counter (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cells (1×10³) were seeded into a 96-well plate and exposed to ivermectin (0.25, 0.5, 1, 2, 4 and 8 µM) or paclitaxel (0.1, 0.1, 1, 10 and 100 nM) for 72 h, or their respective vehicles, ethanol or DMSO.

MDA-MB-231 cells (5×10⁵) were seeded in 75 cm² culture flasks and incubated in complete medium for 48 h to achieve a confluence of ~90%. Cells were washed once with PBS and then harvested with 0.05% trypsin + 0.025% EDTA. Detached cells were washed with blocking reagent (1% FBS in PBS) and resuspended in 1 ml wash buffer (1×10⁶ cells/100 µl), containing the fluorophore-conjugated monoclonal antibodies against CD44 (fluorescein isothiocyanate-conjugated; catalog no. 555742; BD Biosciences, Franklin Lakes, NJ, USA) and against human CD24 (phycoerythrin-conjugated; catalog no. 311106; BioLegend, Inc., San Diego, CA, USA) or their respective isotype controls (FITC-IgG2bκ, catalog no. 555742; PE-IgG2aκ, catalog no. 555574; BD Biosciences). Antibodies were used at the manufacturer's recommended concentrations (dilution: 20 µg/1×10⁶ cells in 100 µl) and incubated at 4°C in the dark for 40 min. Labeled cells were washed in the wash buffer twice. The stem cell marker profiling analysis was performed using a BD FACScanto II (BD Biosciences). Live cell sorting was performed using a BD FACSAria (BD Biosciences). The percentage of cells in different marker populations was evaluated using BD FACSDiva version 6.1.3 (BD Biosciences). The sorted cells were resuspended three times in DMEM-F12-10% FBS with 10,000 U/ml penicillin, 10 mg/ml streptomycin and 0.025 mg/ml amphotericin B. Cells were counted using the trypan blue method and seeded (1×10³ cells/well) in a 96-well plate with an ultra-low attachment surface (Corning-Costar; Corning Inc.) in Human MammoCult^™ Proliferation supplement (STEMCELL Technologies, Inc.), 0.48 µg/ml hydrocortisone and 4 µg/ml heparin, for 24 h at 37°C in humidified atmosphere of 5% CO₂. Cells were treated with ivermectin (0.25, 0.5, 1, 2, 4 or 8 µM) or paclitaxel (0.1, 0.1, 1,10 or 100 nM) or their respective vehicle controls for 72 h. Finally, cells were stained with 0.4% trypan blue to assess cell viability and counted with TC10 Automated Cell Counter (Bio-Rad Laboratories, Inc., Hercules, CA, USA) as aforementioned.

Unsorted, whole population MDA-MB-231 cells were treated with ivermectin for 72 h and total RNA was isolated when cells reached ~70% confluence, using TRIzol reagent (Gibco; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. RNA purity and integrity were assessed by spectrophotometric analysis using a NanoDrop 2000c (Thermo Fisher Scientific, Inc.) and denaturing 2% agarose gel; bands were visualized using a MiniBIS Pro D-Transilluminator (DNR Bio-Imaging Systems Ltd., Neve Yamin, Israel).

A total of 1 µg total RNA was used for cDNA synthesis with the GeneAmp RNA PCR Core kit (Applied Biosystems; Thermo Fisher Scientific, Inc.). cDNA was used with iQ SYBR Green SuperMix (Bio-Rad Laboratories, Inc.), according to the manufacturer's protocol. qPCR reactions were run in triplicate using an ABI Prism 7000 (Applied Biosystems; Thermo Fisher Scientific, Inc.). The qPCR cycling conditions were as follows: 10 min at 95°C; 40 cycles of 30 sec at 95°C, 30 sec at 60°C and 30 sec at 72°C. Data were analyzed using the 2^−ΔΔCq method (12), and reported as the fold-change in gene expression normalized to the endogenous control gene hypoxanthine phosphoribosyltransferase 1 (HPRT1), and relative to cells without treatment. The primers used were: HPRT1 forward, 5′-GAACCTCTCGGCTTTCCCG-3′ and reverse, 3′-CACTAATCACGACGCCAGGG-5′; homeobox protein nanog (nanog) forward, 5′-ACCTCGCTGATTAGGCTCCAA-3′ and reverse, 3′-AGTCTGGACACTGGCTAATCC-5′; octamer binding protein 4 (oct-4) forward, 5′-CAGGCCCGAAAGAGAAAGC-3′ and reverse, 3′-CCACACTCGGACCACATCCT-5′; and sox-2 forward, 5′-GCTAGTCTCCAAGCGACGAAA-3′ and reverse, 3′-AATTCAGCAAGAAGCCTCTCCTT-5′. The annealing temperature was 60°C for all reactions.

Unsorted whole population MDA-MB-231 cells (4×10⁵) were cultured in 25 cm² flasks and treated with ivermectin (or its vehicle control) for 72 h. Once the cells had reached a density of ~70%, the cells were washed once with PBS and then harvested with 0.05% trypsin/0.025% EDTA. Detached cells were washed with PBS, proteins were extracted with radioimmunoprecipitation buffer (150 mM NaCl; 1.0% IGEPAL CA-630; 0.5% sodium deoxycholate; 0.1% SDS; 50 mM Tris, pH 8.0) in the presence of proteinase inhibitors (catalog no. p8340; Sigma-Aldrich; Merck KGaA). Protein concentration was determined using a bicinchoninic acid assay and the integrity was assessed by coomassie staining. A total of 30 µg protein was separated by 10% SDS-PAGE and transferred onto a polyvinylidene difluoride membrane (cat no. 162-0177; Bio-Rad Laboratories, Inc.). The membrane was blocked with 3% skim milk in PBS for 1 h at room temperature and subsequently incubated with antibodies against nanog (catalog no. sc-134218; 1:500); sox-2 (catalog no. sc-17320; 1:200); oct-4 (catalog no. sc-9081; 1:200) (all from Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-actin peroxidase (A3854; 1:10,000; Sigma-Aldrich; Merck KGaA) in blocking solution (5% skim milk in TBS + 0.1% Tween-20), overnight at 4°C. The following secondary antibodies were used: Oct4, bovine anti-rabbit, sc-2370; sox-2, donkey anti-goat, sc-2020; nanog, anti-mouse, sc-2371 (all from Santa Cruz Biotechnology, Inc). Secondary antibodies were diluted 1:1,000 and the incubation was performed for 1 h at room temperature. Protein bands were visualized using the chromogenic substrate Clarity Western Enhanced Chemiluminescence Substrate (catalog no. 1705060; Bio-Rad Laboratories, Inc.). Bands were quantified densitometrically using Image J version 1.50f (National Institutes of Health, Bethesda, MD, USA).

Analyses were performed with GraphPad Prism software (version 6.0; GraphPad Software, Inc., La Jolla, CA, USA). The experiments were conducted with at least three independent triplicates. P-values were calculated using the one-way analysis of variance test followed by the Bonferroni post-hoc test. P<0.01 was considered to indicate a statistically significant difference.

Compounds identified in DrugBank possessed a low molecular similarity with salinomycin (median and mean similarity values of 0.34±0.14). Subsequent results from the similarity searching using salinomycin as a query molecule identified 25 top-ranked compounds with a high similarity value. To define a compound with ‘high’ similarity value, molecules were selected as those with similarity values two standard deviations above the mean similarity of all DrugBank compounds to salinomycin. Following visual inspection of the chemical structures of the identified compounds, the drugs with the highest similarity scores were ivermectin followed by natamycin (a food preservative) and narasin (an antibiotic for veterinary use). Ivermectin had the highest similarity value of 0.78, and following the principle of molecular similarity, which states that similar compounds demonstrate similar properties (13) (with the exception of the so-called activity cliffs) (14), it was hypothesized that the antiparasitic drug ivermectin may also possess similar biological properties as salinomycin. In addition, ivermectin is a Food and Drug Administration (FDA)-approved drug for clinical use, whereas natamycin and narasin are not. Notably, using this method of screening that is based on structural similarity rather than biological activity upon CSC cells, as reported by Gupta et al (7), did not identify avermectin as a candidate compound. Two- and three-dimensional structures of salinomycin and ivermectin are provided in Fig. 1.

To evaluate the antitumoral effects of ivermectin on MDA-MB-231 cells, cell viability and clonogenicity was evaluated. A mild-to-moderate dose-dependent effect was observed in cells treated with various concentrations of ivermectin (Fig. 2A); however, cell viability did not decrease beyond 45%, even at 8 µM of ivermectin. Statistically significant differences were observed at concentrations between 0.8 and 8 µM, compared with untreated controls (P<0.0001). Colony forming ability was also significantly reduced with ivermectin treatment with doses as low as 0.2 µM (Fig. 2B); however, these effects appear to be similar at doses between 1 and 8 µM (P<0.0001).

The CD44⁺/CD24⁻ subpopulation of MDA-MB-231 cells has been previously reported to possess stem/progenitor cell properties, and this subpopulation in patients with breast cancer exclusively retains the ability to form novel tumors in a non-obese diabetic-severe compromised immunodeficiency mouse xenograft model (15,16). The proportion of CD44⁺/CD24⁻ cells in the cell line MDA-MB-231 is 85±5% (17). In addition, spheroids of cell lines growing in nonadherent conditions are also reported to be enriched in stem cells (18). Therefore, these two conditions were used to determine whether ivermectin treatment preferentially inhibited the CSC population compared with treatments with the cytotoxic drug paclitaxel. At concentrations ≤2 µM ivermectin, there were no significant differences in viability in the total MDA-MB-231 population compared with cultures of cells grown in spheroids and those sorted by CD44⁺/CD24⁻ (Fig. 3A). However, cells treated with 4 or 8 µM ivermectin exhibited a significant decrease in viability in the CSC-enriched populations compared with the total cell population (P<0.001); whereas viability in the total cell population was inhibited by ≤30%. As expected, the two subpopulations (spheroids and CD44⁺/CD24⁻) exhibited less inhibition with paclitaxel treatments compared with ivermectin treatments, and no significant differences were identified between either of these groups compared with the inhibition exhibited in the total MDA-MB-231 cell population (Fig. 3B). Ivermectin treatment exhibited an increased inhibitory effect upon CSC-enriched subpopulations compared with cells treated with paclitaxel.

To further evaluate the relative selectivity of ivermectin upon the stem cell population, CD44⁺/CD24⁻ and CD44⁺/CD24⁺ subpopulations of MDA-MB-231 cells were sorted and cultured in the presence of either ivermectin or paclitaxel. Cells treated with 4 and 8 µM ivermectin exhibited a significant reduction in viability of the CD44⁺/CD24⁻ stem cell population; 8 µM ivermectin treatment reduced the CD44⁺/CD24⁻ viability to 0%, compared with CD44⁺/CD24⁺ cells (Fig. 4A), whereas the reduction in viability in CD44⁺/CD24⁺ cells treated with 8 µM ivermectin approached 25%. The non-CSC CD44⁺/CD24⁺ cell populations were sensitive to paclitaxel from 0.01 nM; whereas inhibition of the CD44⁺/CD24⁻ stem cell subpopulation was only observed at ≥10 nM. Statistical differences were observed at all concentrations between CD44⁺/CD24⁺ and CD44⁺/CD24⁻ (Fig. 4B).

As salinomycin has been demonstrated to decrease the expression of stemness genes including nanog (19), MDA-MB-231 cells cultured in normal (adherent) conditions were treated with ivermectin at 4 µM for 72 h and the mRNA expression levels of nanog, sox-2 and oct-4 were evaluated by RT-qPCR and western blotting. As demonstrated in Fig. 5, ivermectin significantly reduced the expression of these three genes at both the mRNA and the protein level (P<0.01).