KYS05090S (molecular weight, 626.69 g/mol; molecular formula, C37H45Cl2N5O) (10,14) was synthesized and supplied by Professor Jae Yeol Lee (Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung Hee University, Seoul, Korea; Fig. 1A). Interleukin-6 (IL-6) was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Ribonuclease A (RNase; cat. no. R6513) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). DC Protein Assay kit II (cat. no. 500-0113) for the protein quantification during Western blotting was purchased from Bio-Rad Laboratories, Inc. (Hercules, CA, USA). DMSO, propidium iodide (PI), bovine serum albumin (BSA) and formaldehyde solution were obtained from Sigma-Aldrich (Merck KGaA).

Ovarian cancer cell lines SKOV3 (cat. no. ATCC^® HTB-77™), OVCAR3 (cat. no. ATCC HTB-161™) and HEK293 cells (cat. no. ATCC CRL-1573™) were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic (Welgene, Inc., Gyeongsan, South Korea) at 37°C in a humid environment containing 5% CO₂.

To evaluate the cytotoxicity of KYS0590S, SKOV3 and OVCAR3 ovarian cancer, and HEK293 cells were seeded onto 96-well tissue culture plates (1×10⁴ cells/well) and were exposed to various concentrations (0, 1.5, 3, 6 and 12 μM) of KYS0590S at 37°C for 24 h. Subsequently, MTT solution (1 mg/ml) was added to each well for 1-2 h at 37°C in the dark and dimethyl sulfoxide was then added to each well at room temperature for 10 min. Optical density (OD) was measured using a microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA) at a wavelength of 570 nm. Cell viability was calculated as a percentage of viable cells in the KYS0590S-treated group, compared with the untreated control.

DNA fragmentation was analyzed using an In Situ Cell Death Detection kit (Roche Diagnostics, Basel, Switzerland). SKOV3 and OVCAR3 cells (4×10⁴ cells/well) were plated onto 4 well Chamber slides (SPL Life Sciences, Pocheon, South Korea) and exposed to KYS05090S (5 μM) at 37°C for 24 h. The cells were fixed in 4% paraformaldehyde at room temperature for 1 h and treated with TUNEL buffer containing fluorescein isothiocyanate fluorescein-12-dUTP for 1 h at 37°C in the dark. The slides were mounted with mounting medium containing DAPI (Vector Laboratories, Inc., Burlingame, CA, USA) and visualized at ×200 magnification using FLUOVIEW FV10i confocal microscopy (Olympus Corporation, Tokyo, Japan) in three fields of view.

SKOV3 and OVCAR3 cells were treated with KYS05090S (0, 3 and 5 μM) at 37°C for 24 h and washed with PBS three times. Ethanol (70%) was used to fix the cells at 4°C overnight. The cells were treated with RNase (1 mg/ml) at 37°C for 1 h, stained with PI at 37°C for 1 h and then quantified using a FACS Caliber flow cytometer and CellQuest software Version 5.2.1 (BD Biosciences; Becton Dickinson and Company, Franklin Lakes, NJ, USA).

SKOV3 and OVCAR3 cells were exposed to KYS05090S (3 and 5 μM) at 37°C for 24 h. The ovarian cells were fixed in 4% (v/v) methanol-free formaldehyde solution at room temperature for 1 h. The slides were mounted, and nuclei were counterstained with DAPI (10 μg/ml; Sigma-Aldrich; Merck KGaA) solution for 25 min at room temperature in the dark, and were visualized at ×40 and ×63 magnification by Zeiss LSM700 confocal microscopy.

SKOV3 cells (5×10⁴ cells/well) were plated onto cell culture chamber slides (SPL Life Sciences) following serum-starvation (DMEM) at 37°C for 3 h. The cells were treated with KYS05090S (5 μM) at 37°C for 24 h and IL-6 (30 ng/ml) at 37°C for 15 min, and then fixed in 4% (v/v) methanol-free formaldehyde solution (pH 7.4) at 4°C for 25 min. Following permeabilization in 0.2% (v/v) Triton X-100, the cells were blocked at room temperature for 1 h with 5% (v/v) BSA, 0.5% (v/v) Tween-20 in PBS and incubated with STAT3 antibody (cat. no. 12640; Cell Signaling Technology, Inc., Danvers, MA, USA; 1:1,000) at 4°C overnight. Anti-mouse IgG Alexa Fluor 488 (cat. no. A28175; Thermo Fisher Scientific, Inc., Waltham, MA, USA; 1:1,000) was used as the secondary antibody and incubated for 1 h at room temperature. The slides were mounted using mounting medium (Vector Laboratories, Inc.) and nuclei were counterstained with DAPI at room temperature for 15 min. Subsequently, the slides were visualized under a FLUOVIEW FV10i confocal microscope (Olympus Corporation).

SKOV3 and OVCAR3 cells were lysed in Radioimmunoprecipitation assay buffer (50 mM Tris-HCl, 150 mM NaCl, 2 mM EDTA and 1% Triton X-100) containing protease inhibitors (Roche Diagnostics GmbH, Mannheim, Germany), and phosphatase inhibitors (Sigma-Aldrich; Merck KGaA). Protein samples (100 μg) were centrifuged at 15,000 × g and 4°C for 30 min and the supernatant was distributed into 96-well plate, exposed to the solutions of a DC protein assay kit at room temperature for 10 min and then the absorbance was detected at 665 nm with Bio-Rad model 680 ELISA reader. The protein was separated by SDS-PAGE on an 8% gel and an equal amount of protein was loaded on SDS-PAGE and transferred to nitrocellulose membranes. Membranes were blocked by 5% skim-milk (diluted in TBST) solution at room temperature for 1 h. Membranes were incubated with primary antibodies to detect cleaved-poly(ADP-ribose) polymerase (PARP; cat. no. 9542; 1:1,000), cleaved-caspase-3 (cat. no. 9664; 1:1,000), cleaved-caspase-9 (cat. no. 9505; 1:1,000), cyclin D1 (cat. no. 2978; 1:1,000), c-Myc (cat. no. 5605; 1:1,000), phospho (p)-JAK2 (cat. no. 3776s; 1:1,000), JAK2 (cat. no. 3230s; 1:1,000), p-STAT3 (cat. no. 9145; 1:1,000) and STAT3 (cat. no. 12640s; 1:1,000; all Cell Signaling Technologies, Inc.), B-cell lymphoma-2 (Bcl-2; cat. no. sc-492; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and β-actin (cat. no. A2228; 1:10,000; Sigma-Aldrich; Merck KGaA) diluted in PBS-Tween-20 with 3% BSA overnight at 4°C, washed three times with 0.2% PBS-Tween-20, and incubated with horseradish peroxidase-conjugated secondary anti-rabbit (cat. no. 7074; 1:5,000; Cell Signaling Technologies, Inc.) and anti-mouse (cat. no. sc-516102; 1:10,000; Santa Cruz Biotechnology, Inc.) IgG antibodies at 4°C overnight. The blot expression was visualized with enhanced chemiluminescence (GE Healthcare Life Sciences, Little Chalfont, UK). Additionally, SKOV3 cells were exposed to IL-6 at 4°C for 15 min to activate the JAK2/STAT3 pathway.

Data are presented as the mean ± standard deviation. All experiments were repeated at least three times. The statistically significant differences between control and KYS05090S treated groups were determined using one-way analysis of variance and Tukey post hoc test. P<0.05 was considered to indicate a statistically significant difference by using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA).

The cytotoxicity of KYS05090S (Fig. 1A) was evaluated in SKOV3 and OVCAR3 ovarian cancer cells, and HEK293 cells. KYS05090S notably increased cytotoxicity in a concentration-dependent manner in SKOV3 and OVCAR3 cancer cells; whereas, it exhibited comparatively reduced cytotoxicity in normal HEK293 cells (Fig. 1B).

Subsequently, to determine the apoptotic effect of KYS05090S, DAPI staining and a TUNEL assay were conducted in SKOV3 and OVCAR3 cells. Nuclei fragmentation, abnormal margins and condensed chromosomes were observed in KYS05090S-treated SKOV3 and OVCAR3 cells (Fig. 2A). To further investigate the KYS05090S-induced apoptosis, cell cycle analysis was performed, since the sub-G1 phase population exhibits DNA fragmentation and loss due to apoptosis (15). As expected, KYS05090S dose-dependently increased the sub-G1 cell population in SKOV3 and OVCAR3 ovarian cancer cells (Fig. 2B-D). Similarly, a TUNEL assay demonstrated that KYS05090S increased the number of TUNEL-positive SKOV3 and OVCAR3 cells, compared with untreated control cells (Fig. 2E).

To investigate the apoptotic mechanism of KYS05090S, western blot analysis was performed to determine the expression of pro- and anti-apoptotic proteins in SKOV3 and OVCAR3 cells. In the present study, KYS05090S treatment increased the cleavage of PARP, and activated caspase-9 and caspase-3 in the ovarian cancer cells, compared with untreated control cells (Fig. 3). Consistently, KYS05090S decreased the expression of anti-apoptotic proteins, including cyclin D1, Bcl-2 and Bcl-extra large (Bcl-xl) in OVCAR3 and SKOV3 cells (Fig. 4). Additionally, the level of p-STAT3 was significantly (P<0.05) attenuated by KYS05090S treatment in both ovarian cancer cell lines by KYS05090S treatment. KYS05090S treatment also dose-dependently reduced the level of p-JAK2 and c-Myc in SKOV3 cells (Fig. 4B).

Subsequently, the role of JAK2/STAT3 signaling in KYS05090S-induced apoptosis in SKOV3 cells was investigated, as JAK2/STAT3 signaling is involved in cancer cell proliferation and progression (16). It is well established that IL-6 activates JAK2/STAT3 signaling (16-18). In the present study, p-JAK2 and p-STAT3 levels were increased by 30 ng/ml IL-6 treatment and the phosphorylation effect was maximal at 15 min after IL-6 treatment (Fig. 5A). Therefore, SKOV3 cells were exposed to IL-6 for 15 min to activate the JAK2/STAT3 pathway in the subsequent experiments. KYS05090S treatment significantly suppressed the IL-6-induced phosphorylation of JAK2 and its downstream mediator, STAT3, in SKOV3 cells (P<0.05; Fig. 5B).

Furthermore, nuclear translocation is essential for the anti-apoptotic function of STAT3 within the JAK2/STAT3 pathway (19). Therefore, immunofluorescence staining for STAT3 and PI staining were performed to observe nuclear translocation of STAT3 in SKOV3 cells. IL-6 treatment successfully induced STAT3 nuclear translocalization in SKOV3 cells (P<0.05; Fig. 6). However, KYS05090S treatment significantly reversed the increased nuclear translocation of STAT3 induced by IL-6, compared with untreated control cells.