Tissue was washed twice in cold phosphate buffered saline before being minced into 5 mm pieces in a sterile culture dish. The minced pieces were transferred into 50 mL conical tubes containing Hanks' balanced salts (Sigma-Aldrich, St Louis, MO, USA), supplemented with 25 mM HEPES, 100 units/mL antibiotics, 1.5 mg/mL collagenase IV (Sigma-Aldrich) and 0.2 mg/mL of DNase I (Roche Diagnostics, Mannheim, Germany). All the tubes were kept at 37℃ in a water bath with gentle agitation for 3 hours. Undigested tissue was filtered and the cells were centrifuged at 1,000 rpm for 5 minutes. The pellet was rinsed once with Hanks' balanced salts and dispersed in complete medium composed of DMEM/F12 with 10% fetal bovine serum, 1% penicillin/streptomycin. The culture medium was changed every two days.

The number of viable cells was evaluated by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Initially, cells were seeded at a density of 1×105 cells/mL in 24-well plate, and then cultured to allow their adhesion to the plate. After this pre-incubation, the culture medium was changed to experimental medium supplemented with salinomycin 1, 5, 10, and 20 µM or DMSO (control) for 24 hours. MTT reagent was added and incubated for an additional 3 hours at 37℃. The intensity of the purple color formed in this assay is proportional to the number of viable cells. Optical density (OD) was measured at 540 nm. Cell survival was calculated by subtracting the background OD of media alone and then normalized by dividing the OD of test wells by the OD of control (untreated) wells. The mean value and their standard deviation were calculated from triplicate experiments.

Salinomycin treated cells were harvested in 1×RIPA buffer (Thermo Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitors with EDTA (Thermo Scientific). 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 by Semi-Dry Transfer Cell (Bio-Rad). The membrane was blocked with Tris-buffered saline containing 5% skim milk. After washing, the membranes were incubated with primary antibodies to Akt, pAkt, cleaved caspase-3, cleaved caspase-8, cleaved caspase-9 and c-PARP (Cell Signaling, Beverly, MA, USA), Bcl-2, Fas, and DR4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), DR5 (Koma Biotech, Seoul, Korea), Bax and XIAP (BD Pharmingen, Franklin Lakes, NJ, USA), FLICE-like inhibitory protein (c-FLIP; Enzo Life Science International, Farmingdale, NY, USA) and β-actin (Santa Cruz Biotechnology). After reaction with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology), bands on the membranes were visualized by an enhanced chemiluminescence (ECL) system (Thermo Scientific). The density of respective bands was analyzed by the Chemi-doc XRS imaging system (Bio-Rad).

After drug treatment, cells were centrifuged at 2,000 rpm for 5 minutes to harvest. Cells (5×106) were then lysed in 50 µL of lysis buffer on ice for 30 minutes and centrifuged at 14,000 rpm for 5 minutes at 4℃ and the supernatant was 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℃ for 2 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, Palo Alto, CA, USA).

Fragmented nucleosomal DNA was quantified by cell death detection enzyme-linked immunosorbent assay (ELISA) plus kit (Roche Diagnostics) as described in the manufacturer's manual. Briefly, cells (4×105) were plated in 6 well plates in the presence of 10% fetal bovine serum with antibiotics (streptomycin and penicillin). The cells were exposed to 0, 5, 10 µM salinomycin for 24 hours. After careful removal of media, cells were pooled and lysed with 200 µL of lysis buffer for 30 minutes at room temperature. Lysates were transferred to 1.5 mL eppendorf tubes and centrifuged at 13,000 rpm for 10 minutes to obtain low molecular weight DNA from apoptotic cells. Supernatant (20 µL) was used to detect apoptosis with microplate reader (Tecan Sunrise, Durham, NC, USA) at 405 nm. Background values (incubation buffer alone) were subtracted and OD values representing nucleosomal DNA fragments in salinomycin treated samples were compared with those values obtained from control cells and expressed as percentage (%) of control.

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

Author used an MTT assay to analyze the effect of salinomycin on the growth of uterine leiomyoma cells. As shown in Fig. 1, in contrast with the control, cells treated with salinomycin showed decreased cell growth in a dose-dependent manner. The leiomyoma cell growth was decreased by about 95%, 73%, 62%, and 30% in the treatment with salinomycin 1, 5, 10, and 20 µM, respectively in contrast with the untreated control (Fig. 1).

The induction of apoptotic cell death could be in part due to inactivation of important survival genes and therefore the expression of molecules transcriptionally regulated by Akt was also evaluated. The expression of pAkt proteins were decreased upon salinomycin treatment in uterine leiomyoma cells. Next, we investigated whether apoptosis induced by salinomycin occurs via intrinsic mitochondrial apoptosis pathway in uterine leiomyoma cells. For that purpose, we measured the levels of Bcl-2 family proteins (Bcl-2 and Bax) and XIAP. There was no change in the expression of Bax but the antiapoptotic Bcl-2 protein and XIAP found decreased. The caspase-3 and PARP protein cleavage were increased after treatment (Fig. 2). These results suggest that salinomycin induced cell growth inhibition and induction of apoptotic cell death results in part from the inhibition of survival factors, and inactivation of Akt pathway.

In this step, we investigated whether salinomycin activated the extrinsic apoptosis pathway by measuring the expression of death receptor signaling-related proteins, such as DR4, DR5, Fas and caspase-8. The expression of DR5, Fas and cleaved caspase-8 proteins were significantly increased. In addition, we observed down-regulation of c-FLIP expression. There was no change in the expression of DR4 (Fig. 3). These results suggest that salinomycin-induced apoptotic cell death involve the activation of DR and Fas mediated pathway.

The apoptosis assay was performed to evaluate the mechanism of the inhibition of using uterine leiomyoma cell growth using csapase-3 activity test and cell death detection kit. Activity of caspase-3 was increased following salinomycin treatment (Fig. 4A). ELISA showed significant increase of histone associated DNA fragments after 24 hours exposure to salinomycin (Fig. 4B).