HeyA8, CAOV3, OVCAR3 and SKOV3 ovarian cancer cell lines derived from humans were used. CAOV3 (cat. no. 30075), OVCAR3 (cat. no. 30161) and SKOV3 (cat. no. 30077) cells were purchased from the Korean Cell Line Bank. CAOV3, OVCAR3 and SKOV3 cells were derived from a human ovarian adenocarcinoma. HeyA8 cells were a kind gift from a laboratory of the Department of Systems Biology, MD Anderson Cancer Center, Houston, TX, USA. The HeyA8 cells originated from a human ovarian cancer xenograft (HX-62) that was developed from a peritoneal metastatic mass of a patient with moderately differentiated papillary cystadenocarcinoma of the ovary (13).

All cancer cell lines were maintained in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) containing 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), streptomycin (10,000 µg/ml), penicillin (10,000 U/ml) and amphotericin B (25 µg/ml) at 37°C in humidified atmosphere with 5% CO₂. For the 3D culture, each well of a 96- or 12-well plate were coated with thawed Matrigel (Growth Factor Reduced Matrigel; Corning Inc.) and seeded with ovarian cancer cells. A total of 1×10⁴ HeyA8, CAOV3, and OVCAR3 cells/well were seeded in a 12-well plate coated with Matrigel, and the 3D structures attained 80% confluence after 4 days of incubation. During the FDI-6 (Axon Medchem) treatment, the FBS content of the medium was changed to 2%. The plates were kept inside an incubator at 37°C for 24 h for western blot analysis, cell viability assay and RPPA analysis. Inhibitors of proteins involved in the compensatory signaling pathway were simultaneously supplied with FDI-6 to the ovarian cancer cells. N-Ras, phosphoprotein kinase C-delta (p-PKCδ) (S664), and HER3 were selected as proteins involved in the compensatory adaptive response. The present study used tipifarnib (Cayman Chemical Company) for N-Ras inhibition, rottlerin (Abcam) for p-PKCδ (S664) inhibition and sapitinib (Selleck Chemicals) for HER3 inhibition. Tipifarnib, a farnesyl transferase inhibitor interferes with Ras function by inhibition of farnesyl transferase, the enzyme coupling a 15-carbon isoprenyl group to Ras proteins (14). Rottlerin is a protein kinase inhibitor and inhibits PKCδ by interacting with its ATP-binding site (15). Sapitinib is an EGFR/ErbB family inhibitor, which induces apoptosis in the sensitive cell lines and suppresses phosphorylated (p)-EGFR and its downstream pathways in a dose-dependent manner (16). HER3 possesses impaired kinase activity and preferably heterodimerizes with HER2. HER3-HER2 heterodimerization activates downstream signaling pathways that regulate several cellular processes including cell proliferation, cell survival, apoptosis, tumor growth, and metastasis (17).

Cell viability was analyzed using Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies, Inc.) according to the manufacturer's instructions. A total of 1×10³ HeyA8 and CAOV3 cells and 1×10⁴ SKOV3 cells were seeded into a 96-well plate for the cell viability assay. FDI-6 was added to each cell culture on day 4. The culture medium was removed and 100 µl fresh medium containing 10 µl CCK-8 solution was added to each well. The cells were then placed in a 37°C incubator for 4 h. The optical density values were determined in triplicate at a wavelength of 450 nm. The cell viability was analyzed in ovarian cancer cells after treatment with FDI-6 alone (0, 3 and 10 µM) with one inhibitor of N-Ras, p-PKCδ (S664), and HER3 (0, 3 and 10 µM) and with the combination of FDI-6 and an inhibitor.

RPPA analysis was performed to identify proteins involved in the compensatory adaptive response induced by FDI-6 in HeyA8 cells. RPPA analysis is a high-throughput method that allows the measurement of protein expression levels in a large number of samples simultaneously using >200 validated antibodies. A total of 1×10⁴ HeyA8 cells/well were seeded in a 12-well plate for RPPA analysis. FDI-6 was added to each cell culture on day 6. HeyA8 cells were treated with FDI-6 at concentrations of 1, 10 and 20 µl on day 6. All tests were performed in duplicate. After thawing Matrigel by adding 800 µl 1X Hank's balanced salt solution with 5 mM ethylenediaminetetraacetic acid to each well, the cells were lysed by mixing 30–100 µl lysis buffer [1% Triton X-100, 50 mM HEPES (pH 7.4), 150 mM NaCl, 1.5 mM MgCl₂, 1 mM EGTA, 100 mM NaF 10 mM Na pyrophosphate, 1 mM Na₃VO4, 10% glycerol] with the cell pellet followed by centrifugation at 18,470 × g and 4°C for 10 min. The bicinchoninate method was used to measure protein concentration. A total of 10 µl SDS sample buffer [40% glycerol, 8% SDS, 0.25 M Tris-HCl (pH 6.8) and β-mercaptoethanol) was added to 30 µl supernatant and heated to 95°C for 5 min. Protein lysate concentrations were adjusted to 1 µg/µl, and a serial dilution of five concentrations was prepared, with 10% of the samples replicated for quality control (2470 Arrayer; Aushon Biosystems Inc.) on nitrocellulose-coated slides (Grace Bio-Labs). Immunostaining was performed using a DakoCytomation-catalyzed system (Dako; Agilent Technologies, Inc.) and diaminobenzidine colorimetric reaction. Spot intensities were analyzed and quantified using MicroVigene software (version 3.0, VigeneTech Inc.) to generate spot signal intensities. Data from the RPPA analysis were rearranged by the rank sum score method and are expressed in a heat map in which green indicates downregulation and red indicates upregulation of protein expression. The heat map was generated using Cluster 3.0 (http://bonsai.hgc.jp/~mdehoon/software/cluster/software.htm) as a hierarchical cluster using Pearson's correlation coefficient and a center metric.

A total of 1×10⁴ HeyA8, CAOV3, and OVCAR3 cells/well and 1×10⁵ SKOV3 cells/well were seeded in a 12-well plate for western blot analysis. FDI-6 was added to each cell culture on day 4. Ovarian cancer cell lysates were prepared using RIPA buffer (Thermo Fisher Scientific, Inc.). Protein concentration was determined through the Lowry method using a DC protein assay kit (Bio-Rad Laboratories, Inc.) following the manufacturer's protocol. Protein samples (5 µg) were separated using 8–12% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Amersham; Cytiva). The membrane was pre-incubated with 5% skimmed milk in TBS for 1 h at room temperature. Membranes were incubated with primary antibodies against FOXM1 (cat. no. 5436; Cell Signaling Technology, Inc.), HER3 (cat. no. 12708; Cell Signaling Technology, Inc.), N-Ras (cat. no. sc-31; Santa Cruz Biotechnology, Inc.), p-PKCδ (S664) (cat. no. sc-365463; Santa Cruz Biotechnology, Inc.), PKCδ (cat. no. ab18216; Abcam), and β-actin (cat. no. A5441; Sigma-Aldrich; Merck KGaA) were diluted 1:1,000 in TBS with 0.1% Tween-20 (TBS-T) overnight at 4°C and then washed three times with TBS-T. Following which, membranes were incubated with HRP-conjugated goat anti-rabbit IgG antibody (1:5,000; cat. no. BR170-6515; Bio-Rad Laboratories, Inc.) or goat anti-mouse IgG antibody (1:5,000; cat. no. BR170-6516; Bio-Rad Laboratories, Inc.) for 1 h at room temperature. The membrane was washed in TBS-T, and hybridized bands were detected using an ECL clarity detection kit (Bio-Rad Laboratories, Inc.) and ChemiDoc XR analyzer software Image Lab 5.1 (Bio-Rad Laboratories, Inc.).

HeyA8 cells were grown in 100-mm dishes to 80% confluence prior to FDI-6 (1, 3 and 10 µM) or 0.1% DMSO control treatment for 24 h. For chromatin isolation, cells were harvested using a chromatin extraction kit (Abcam) according to the manufacturer's protocol. First, cells were cross-linked with 1% formaldehyde in 10 ml culture media by rocking at 50 rpm for 10 min at room temperature and added to 1.1 ml of 1.25 M glycine. Subsequently, cells were harvested by scraping into cold PBS and centrifuged at 20,500 × g for 5 min at 4°C. Cell pellets were lysed sequentially with lysis buffer and resuspended in extraction buffer containing protease inhibitors. Samples were homogenized on ice using a Q125 sonicator (Qsonica; Misonix, Inc.) for 2 min at medium power with a pulse of 20 sec on/30 sec off and centrifuged at 13,570 × g for 10 min at 4°C. The supernatant was collected and dialyzed in chromatin buffer at a 1:1 ratio. Protein concentration was measured using a DC protein assay kit (Bio-Rad Laboratories, Inc.) according to the manufacturer's protocol. Protein samples (5 µg) were separated using 8–10% SDS-PAGE and further analyzed using western blot analysis. Incubation with primary antibody against FOXM1 (1:1,000), histone H3 (1:5,000; cat. no. ab1791; Abcam) and GAPDH (cat. no. A9545; Sigma-Aldrich; Merck KGaA) was performed overnight at 4°C.

HeyA8 cells were seeded in 12-well plates and treated with 0, 3 and 10 µM rottlerin for 24 h. Cells were washed with cold PBS and total ATP levels were measured in cell lysates using a Colorimetric ATP Assay kit (cat. no. ab83355; Abcam) according to the manufacturer's protocol. Briefly, cells were resuspended in 100 µl ATP assay buffer, centrifuged at 13,000 × g for 5 min at 4°C and protein was removed from the supernatant using Deproteinizing Sample Preparation kit (cat. no. ab204708; Abcam). A total of 40 µl deproteinated samples were added to the ATP reaction mix in 96-well clear plates and incubated at room temperature for 30 min in the dark. The absorbance of samples was measured using a VersaMax microplate reader (Molecular Devices, LLC) at a wavelength of 570 nm and the results were calculated relative to the standard curve.

Images of ovarian cancer cells following treatment with FDI-6 and inhibitors for 24 h were captured under ×100 magnification using an inverted microscopy (CKX41; Olympus Corporation).

One-way ANOVA and Tukey's Honest Significant Difference test was applied to evaluate the differences in cell viability and protein expression. All statistical analyses were performed using SPSS (version 20; IBM Corp.). P<0.05 was considered to indicate a statistically significant difference.

FOXM1 expression was assessed in several ovarian cancer cells in order to select the cells for use in subsequent experiments. FOXM1 expression was the highest in HeyA8 cells based on western blot analysis (Fig. 1A). FOXM1 expression was also observed in CAOV3 and OVCAR3 cells, but the expression level was rather low in SKOV3 cells. Hence, HeyA8 and CAOV3 cells were selected for subsequent experiments and SKOV3 cells were used as controls. FDI-6 inhibited FOXM1 function by displacing FOXM1 from its genomic targets in HeyA8 cells (Fig. 1B). Western blot analysis on chromatin fractions revealed a specific displacement of DNA-bound FOXM1 following treatment with FDI-6. RPPA analysis was performed to identify the proteins associated the adaptive signaling pathway response induced by FDI-6 in HeyA8 cells (Fig. 2). In cells treated with the highest concentration of FDI-6 (20 µM), the concentration of DMSO was 0.1%, and the concentration of DMSO in the control was also 0.1%. When cells were treated with a lower concentration of FDI-6, less DMSO was included and the effect of DMSO on cells was minimized (Fig. 2C). Different concentrations of DMSO may cause an initial decrease in expression of these proteins at low doses of FDI-6. As shown in Fig. 2A and B, proteins with a high expression level after inhibition of FOXM1 are displayed in red in the upper part of the heat map, and proteins with low or no expression are shown in green in the lower part of the heat map. In the heat map, the proteins are arranged according to the degree of expression from top to bottom. Proteins with a high expression level (in red) and associated with the cell signaling pathway were chosen first. Bak and Bcl2 are apoptotic proteins and were excluded from candidate proteins. Validation by western blot analysis identified the proteins with different expression levels from those shown in RPPA analysis, and these were also excluded. Smad4 was shown to be differently expressed on western blot analysis and was excluded (data not shown). After these processes, N-Ras, p-PKCδ (S664) and HER3 were chosen as candidate proteins and PKCδ was added to normalize the signals obtained using p-PKCδ (S664) (Fig. 3).

Increased N-Ras expression induced by FDI-6 in HeyA8 cells was weakened in CAOV3 cells, and p-PKCδ (S664) and HER3 even showed decreased levels compared with HeyA8 cells (Fig. 3B). CAOV3 cells showed lower expression of FOXM1 (Fig. 1A) compared with HeyA8 cells and may be one of the reasons for differences in the expression of N-Ras, p-PKCδ (S664) and HER3 between HeyA8 and CAOV3 cells. Only N-Ras in CAOV3 cells was selected as a candidate protein. Tipifarnib as an N-Ras inhibitor, rottlerin as a p-PKCδ (S664) inhibitor and sapitinib as a HER3 inhibitor at concentrations of 3.33 and 10 µM were treated in HeyA8 cells to validate their ability to inhibit N-Ras, p-PKCδ (S664) and HER3 expression. The combination of FDI-6 with tipifarnib attenuated the upregulation of N-Ras induced by FDI-6 in HeyA8 and CAOV3 cells (Fig. 4A and D). The combination of FDI-6 with sapitinib attenuated p-Akt, which is one of the proteins associated with the HER3 downstream signaling pathway but did not attenuate HER3 expression in HeyA8 cells (Fig. 4B). Sapitinib suppresses p-EGFR and its downstream pathways (16). Rottlerin downregulated p-PKCδ (S664) by inhibiting the activity of the kinase that transfers a phosphate group to PKCδ, and the ATP levels in HeyA8 cells was significantly higher after inhibition of kinase activity in the rottlerin-treated group (Fig. 4C). There were significant reductions in the survival of HeyA8 cells by the supplementation of tipifarnib or rottlerin to FDI-6 treatment compared with a single FDI-6 treatment (Fig. 5A and C). However, the addition of sapitinib did not show a significant reduction in cell viability of HeyA8 cells (Fig. 5B). Unlike in HeyA8 cells, tipifarnib did not induce significant reduction in the cell viability of CAOV3 cells (Fig. 5D). In addition, there was no significant reduction in cell viability in SKOV3 cells, which showed lower FOXM1 expression (Fig. 5E-G). Fig. 6 shows that the cell viability of ovarian cancer cells when combined with FDI-6 treatment vs. treatment with the single agent tipifarnib, sapitinib or rottlerin. The present study also demonstrated the change in cell viability by microscopic imaging analysis of HeyA8, CAOV3 and SKOV3 cells (Fig. 7).