Human epithelial ovarian carcinoma cell lines ES2 (clear cell carcinoma) and SKOV3 (adenocarcinoma) were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Primary ADSCs were isolated from three healthy adult female donors and were identified as described in a previous study (17). All donors provided written informed consent, and the present study was approved by the Ethics Committee of Union Hospital, Tongji Medical College, Huazhong University of Science and Technology (Wuhan, China), approval no. 2014073. Primary ADSCs were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 supplemented with 10% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 mg/ml penicillin and 100 mg/ml streptomycin. The supernatants of ADSCs (passage 5 to 20) cultures were harvested every 48–72 h, and the harvested conditioned medium was centrifuged at 4°C at 300 × g for 5 min, filtered through a 0.22 µm syringe filter and stored at 4°C. ES2 and SKOV3 cells were cultured in DMEM/F12 supplemented with 10% FBS or ADSC-derived conditioned medium for 10 days. All cell lines were cultured at 37°C in a humidified atmosphere containing 5% CO₂.

The effect of ADSC-derived conditioned medium on EOC cells was assessed via the MTT assay. EOC cells were seeded in 96-well plates (Corning Life Sciences, Corning, NY, USA) at a density of 5×10³ cells/well. Following attachment, the cells were treated with various concentrations of cisplatin (0, 10, 30, 60, 90 and 120 µM for ES2 cells; 0, 20, 40, 80, 120, 160 and 200 µM for SKOV3 cells) for 48 h followed by incubation with 20 µl MTT reagent (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) at a final concentration of 0.5 mg/ml at 37°C for 4 h. The medium was removed and 100 µl pure dimethyl sulfoxide was added to each well. Wells without cells containing culture medium were used as blank controls, and wells with cells treated with media without cisplatin served as negative controls. The absorbance was measured at a wavelength of 490 nm using a microplate reader (model iMark-10601; Bio-Rad Laboratories, Inc., Hercules, CA, USA). Cell survival curves were generated, from which the half maximal inhibitory concentration (IC₅₀) values of cisplatin in EOC cells under different culture conditions were derived. Each assay was performed in triplicate.

EOC cells (1×10⁴ cells/well) were seeded in 48-well plates (Corning Life Sciences), exposed to cisplatin (at the IC₅₀ value) at 37°C for 48 h and then fixed with 3.7% paraformaldehyde for 10 min at room temperature. Following washing twice with cold PBS, the cells were stained for 5 min with Hoechst 33342 (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in the dark. Following washing, the cells were observed under a fluorescence microscope (model IX70; Olympus Corporation, Tokyo, Japan). The Hoechst reagent is taken up by nuclei, and the nuclei of apoptotic cells exhibit bright blue fluorescence. Each experiment was repeated three times separately.

ES2 and SKOV3 cells (1×10⁵ cells/well) were seeded in 6-well plates (Corning Life Sciences), grown to confluence and exposed to cisplatin at the IC₅₀ value at 37°C for 48 h. The cells were then collected using trypsin without EDTA, washed twice with PBS. Subsequently, FITC Annexin V Apoptosis Detection kit (catalog no. 556547; BD Biosciences, San Jose, CA, USA) was used to detect apoptosis. According to the manufacturer's protocol, cells were resuspended in binding buffer (FITC Annexin V Apoptosis Detection kit; BD Biosciences), and mixed with Annexin V-fluorescein isothiocyanate and propidium iodide at room temperature in the dark for 15 min. Subsequently, the cells were analyzed by flow cytometry (FACSCalibur; BD Biosciences). Each experiment was repeated at least three times separately, and CellQuest^™ analysis program software, version 5.1 (BD Biosciences) was used to analyze the results.

Total protein from cultured cells was extracted using a radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Haimen, China) containing a protease inhibitor cocktail and quantified using a Bradford assay (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. Proteins (30 µg) were separated by 10% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk for 1 h at room temperature and incubated overnight at 4°C with the following primary antibodies: Rabbit anti-caspase-3 polyclonal antibody (cat no. 9665, 1:1,500 dilution; Cell Signaling Technology, Inc., Danvers, MA, USA) and mouse anti-β-actin polyclonal antibody (cat no. sc-130301, 1:500 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Following washing with Tris-buffered saline containing 0.1% Tween, the membranes were incubated with a horse radish peroxidase-conjugated goat-anti-rabbit (cat no. sc 2004, 1:5,000 dilution) or goat-anti-mouse secondary antibody (cat no. sc-516102, 1:5,000 dilution) (both from Santa Cruz Biotechnology, Inc.) for 1 h at room temperature and visualized using an Enhanced Chemiluminescence system (Beyotime Institute of Biotechnology). The protein bands were quantified using Quantity One v4.62 software (Bio-Rad Laboratories, Inc.). Each experiment was repeated at least three times separately.

For the determination of the intracellular platinum concentration, EOC cells were resuspended in PBS and divided into two equal aliquots: One for protein content measurement and the other for platinum determination. The protein content was measured using the Bradford assay (Beyotime Institute of Biotechnology). For platinum determination, cells were incubated with 0.2% nitric acid at room temperature for 1 h. The intracellular platinum concentrations were measured by flameless atomic absorption spectrometry (AAS; Varian SpectrAA 240FS, Palo Alto, CA, USA) using a standard curve covering a range of 60–240 µg/. The intracellular platinum levels are expressed as ng of platinum per mg of protein. All samples were analyzed three times in duplicate.

All data are expressed as the mean ± standard deviation (n≥3). The statistical significance of differences between two groups was analyzed using a two-tailed Students t-test via SPSS statistical software package, version 16.0 (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate whether ADSCs modulate the sensitivity of EOC cells to cisplatin, ES2 and SKOV3 cells were pretreated with conditioned medium derived from ADSCs for 10 days (named co-cultured ES2 and SKOV3 cells). The sensitivities of the co-cultured cells and their parental cells to cisplatin were then assessed using the MTT assay. As demonstrated in Fig. 1, the co-cultured SKOV3 and ES2 cells exhibited a right-shifted dose-survival curve and became more resistant to cisplatin (Fig. 1). The IC₅₀ values for cisplatin of the co-cultured ES2 and SKOV3 cells were 1.60- and 1.71-fold higher, respectively, than that of the parental cells (Table I). These findings suggested that ADSCs decrease the sensitivity of EOC cells to cisplatin.

To further determine whether ADSCs protect EOC cells against cisplatin-induced apoptosis, co-cultured ES2 and SKOV3 cells and parental cells were exposed to cisplatin for 48 h at their IC₅₀ doses based on the MTT assay. Hoechst 33342 staining was then performed to analyze cisplatin-induced apoptosis. According to fluorescence microscopy images, the co-cultured ES2 and SKOV3 cells revealed significantly lower nuclear fragmentation compared with the parental cells (Fig. 2A and B). Consistently, Annexin V/propidium iodide dual staining followed by flow cytometric analysis demonstrated that ADSC-treated supernatants exhibited a significantly decreased proportion of apoptotic ES2 and SKOV3 cells following exposure to cisplatin. Compared with the parental cells, the proportion of apoptotic cells among the co-cultured ES2 and SKOV3 cells decreased by 1.9- and 2.33-fold, respectively (Fig. 2C-F). In addition, the proportion of co-cultured ES2 and SKOV3 cells in the early phase of apoptosis was 2.14- and 3.01-fold lower, respectively, than the parental cells (Fig. 2E and F). To further explore the molecular mechanisms underlying the ADSC-mediated protection of EOC cells against cisplatin-induced apoptosis, caspase-3 was evaluated by western blot analysis. As presented in Fig. 3, after cisplatin treatment, the expression levels of cleaved caspase-3 in conditioned cells were significantly reduced in the co-cultured ES2 and SKOV3 cells compared with the parental cells. These findings suggested that ADSCs may reduce cisplatin-induced apoptosis in EOC cells by inhibiting caspase-3 activity.

Given that decreased intracellular drug accumulation is a critical mechanism of chemoresistance, the effect of ADSCs on intracellular platinum accumulation in EOC cells was investigated using atomic absorption spectroscopy assays. As presented in Fig. 4, following treatment with cisplatin for 48 h at their IC₅₀ doses, platinum concentrations in the co-cultured ES2 and SKOV3 cells were at least 2.34 and 3.60-fold lower than that the control cells, respectively (Fig. 4). These results suggested that ADSCs may decrease the intracellular platinum concentration in EOC cells.