Ascites was collected from patients diagnosed with advanced stage high-grade serous ovarian carcinoma, after obtaining written informed consent under protocols approved by the Ethical Committee of the Keimyung University Dongsan Medical Center, Korea. Epithelial OCCs were isolated using standard procedures. The ascites fluid was centrifuged at room temperature for 5 minutes at 1,500 rpm. The cell pellets were filtered (100-μm cell strainer) and washed twice with phosphate buffered saline (PBS), and red blood cells (RBCs) were removed by Histopaque-1077 (Sigma-Aldrich, St. Louis, MO, USA). The single cells were dispersed in complete medium composed of DMEM/F12 with 100 units/mL antibiotics. The conditioned medium was changed every two days.

The CD44⁺CD117⁺ cells were sorted from the primary OCCs by using the magnetic-activated cell sorting (MACs; Miltenyi Biotec, Bergisch Gladbach, Germany). CD44⁺ subsets were isolated by using mouse antihuman CD44 antibody coupled to magnetic microbeads (Miltenyi Biotec), followed by the magnetic column selection or depletion. The resulting cells were depleted of CD117⁻ subsets by using mouse antihuman CD117 antibody coupled to magnetic microbeads (Miltenyi Biotec). CD44⁺CD117⁺ cells were named as OCSCs.

The number of viable cell exposed to paclitaxel (PTX) and salinomycin was evaluated by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Initially, cells were seeded in 96-well plate, and cultured for 24 hours to allow their adhesion to the plate. After pre-incubation, the culture medium was changed to experimental medium supplemented with dimethyl sulfoxide (DMSO, 0.1%) control, PTX (10–200 nM) and salinomycin (0.5–5.0 µM) alone or in combination for 48 hours. The intensity of the purple color formed by this assay is proportional to the number of viable cells. MTT reagent was added and incubated for an additional 4 hours at 37°C. The optical density (OD) was measured at 540 nm. The mean values and their standard deviations were calculated from triplicate experiments.

Tumor sphere culture was performed in ultra-low attachment 60 mm dishes (Corning, New York, NY, USA), serum-free DMEM/F12 medium, supplemented with 5 μg/mL insulin (Sigma-Aldrich), 20 ng/mL human recombinant epidermal growth factor (Invitrogen, Carlsbad, CA, USA), 10 ng/mL basic fibroblast growth factor (Invitrogen) and 0.4% bovine serum albumin (Sigma-Aldrich).

Cell (1×10⁷/mL) extracts were prepared in 1×radioimmunoprecipitation assay (RIPA) buffer containing protease and phosphatase inhibitors with EDTA (Thermo Scientific, Rockford, IL, USA). Protein concentrations were measured using Protein Assay Reagent (Bio-Rad, Hercules, CA, USA) following the manufacturer’s protocol. Aliquots of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membrane (Bio-Rad). The membranes were blocked with Tris-buffered saline containing 5% skim milk (Becton Dickinson and Company, Oxford, UK) and 0.2% Tween 20 (Amresco Inc., Solon, OH, USA). The following primary antibodies were used: sex determining region Y-box 2 (SOX2, 1:1,000; Cell Signaling, Danvers, MA, USA), octamer-binding transcription factor 3/4 (OCT3/4, 1:1,000; Cell Signaling), death receptor 5 (DR5, 1:1,000; Koma Biotech Inc., Seoul, Korea), Bcl2-associated X protein (Bax, 1:1,000), B cell lymphoma 2 (Bcl-2, 1:1,000; Biotechnology Inc., Santa Cruz, CA, USA), X-linked inhibitor of apoptosis protein (XIAP, 1:1,000; Becton Dickinson and Company), Survivin (1:1,000; Biotechnology Inc.), and β-actin (1:10,000; Sigma-Aldrich). After reaction with horseradish peroxidase conjugated secondary antibodies (Biotechnology Inc.) bands on the membranes were visualized by an enhanced chemiluminescence system (Thermo Scientific) following the manufacturer’s suggested procedure. The density of respective bands was analyzed by the chemi-Doc XRS imaging system (Bio-Rad).

Transfection of siRNA was performed using ON-TARGETplus (Dharmacon, Lafayette, CO, USA) kit, according to manufacturer’s instruction. SMARTpool sequenced siRNA targeting SOX2 or non-targeting siRNA scrambled control (negative control) was diluted to a working stock solution of 5 μM in RNase free water. Transient transfection was done using the DharmaFECT transfection reagents. After 24 hours of post transfection, cells were used to perform immunobloting and MTT assay.

Annexin V assay was used to detect apoptotic cells by staining cells with both annexin V-fluorescein isothiocyanate (FITC) and PI. Cells were incubated for 48 hours at 37°C with PTX (20 nM) and salinomycin (0.5 µM). After washing with cold PBS, cells were re-suspended in 1×binding buffer. Staining of apoptotic cells was performed using the annexin V-FITC/PI apoptosis assay kit (Becton Dickinson and Company) according to the manufacturer’s instructions. Determination of apoptotic cells was performed in a NovoCyte flow cytometer using NovoExpress 1.0.2 software (ACEA Biosciences, San Diego, CA, USA).

After drug treatment, cells were centrifuged at 2,000 rpm for 5 minutes to harvest. Cells (2×10⁶) were then lysed in 50 µL of lysis buffer on ice for 10 minutes and centrifuged at 14,000 rpm for 5 minutes at 4°C. Collected supernatant (50 µL) was added to an equal volume of 2×reaction/DTT Mix supplemented with DEVD-pNA (50 µM) and incubated at 37°C for 3 hours. Caspase-3 activity was measured as the absorbance at 405 nm of the cleaved substrate pNA followed by ApoAlert Caspase Colorimetric Assay kits User Manual (Clontech Laboratories, Mountain View, CA, USA).

Fragmented nucleosomal DNA was quantified by Cell Death Detection enzyme-linked immunosorbent assay (ELISA) Plus kit (Roche Diagnostics, Mannheim, Germany) as described in the manufacture’s manual. Supernatant (20 µL) was used to detect apoptosis with microplate reader (Tecan, Salzburg, Austria) at 405 nm. Background values were subtracted and OD values representing nucleosomal DNA fragments in Hsp90 inhibitor treated samples were compared with the values obtained from control cells and expressed as percentage (%) of control.

The data are presented as mean±SD. Statistical analysis was conducted using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test for post hoc comparison by SPSS 17.0 (SPSS Inc., Chicago, IL, USA). Statistical significance was set at p<0.05.

The potential of salinomycin to inhibit the growth of OCCs and OCSCs was determined using a MTT assay. In comparison with the control, PTX reduced the viability of OCCs in a dose-dependent manner but only slightly reduced the viability of OCSCs (Fig. 1A). On the other hand, salinomycin reduced the viability of OCSCs in a dose-dependent manner, while only slightly affecting OCCs (Fig. 1B). PTX (20 nM) inhibited the growth of OCCs and OCSCs by 27% and 11%, respectively. Inhibition of growth of OCCs and OCSCs by PTX (20 nM) combined with salinomycin (0.5 µM) was 58% and 59%, respectively (Fig. 1C). These findings suggest that salinomycin and PTX could have a synergistic effect for treating ovarian cancer.

We examined whether salinomycin reduced stemness-related gene and protein expression in OCSCs. After treatment with a combination of PTX and salinomycin, OCSCs down-regulated expression of stemness-related mRNA and protein (Fig. 2A, B) such as SOX2 and OCT3/4. To elucidate the effect of SOX2, we examined OCT3/4 expression following SOX2 siRNA transfection in OCSCs (Fig. 2C). SOX2 silencing sensitized the OCSCs to the anti-cancer effect of PTX (Fig. 2D). Taken together, these data demonstrate that salinomycin affects stemness genes, and that the targeting of SOX2 enhances the effect of PTX on OCSCs.

Spheroid formation has been described as an important feature for the survival of OCSCs [17]. We evaluated the spheroid-forming abilities of OCSCs treated with PTX (20 nM), salinomycin (0.5 µM), a combination of both, and neither (as a control group). OCSCs were grown in nonadherent culture conditions in the absence or presence of salinomycin and PTX for 14 days, and spheroid formation was characterized in vitro (Fig. 3A). PTX-alone treatment reduced 46.6%, and salinomycin-alone treatment reduced 60.0% of the spheroid forming behavior. Cells treated with a combination of salinomycin and PTX showed significant reduction of 86.6% in spheroid formation compared with control cells (Fig. 3B).

We examined whether salinomycin induced apoptosis by measuring the expression of DR5, Bax, Bcl-2, XIAP, and Survivin in OCSCs. The treatment combining salinomycin with PTX significantly increased DR5 and decreased Survivin. There was no change in the levels of Bcl-2, Bax, and XIAP (Fig. 4). These results suggest that salinomycin induced apoptosis through a non-mitochondrial pathway, consequently inhibiting cell growth.

An apoptosis assay was performed to evaluate the cause of the inhibition of OCSC growth using annexin V-FITC/PI staining kit. Flow cytometric analysis showed that the combination of salinomycin and PTX dramatically increased the apoptotic cells upon 48 hours of exposure (Fig. 5A). Under the same conditions, activity of caspase-3, an executioner caspase of apoptosis, showed a 2.25-fold increase, and histone-associated DNA fragmentation showed a 4.67-fold increase (Fig. 5B, C).