SCH was purchased from AdooQ Bioscience. Baze acetate was purchased from Merck KGaA. The stock concentration of SCH and Baze acetate was 20 mM reconstituted in DMSO (Merck KGaA) and stored at −20°C. MTT was purchased from Sigma-Aldrich; Merck KGaA, and a 5-mM stock concentration was prepared in ddH₂O and stored at −20°C. All primary and secondary antibodies were purchased from Cell Signaling Technology, Inc.

SKOV3, CAOV3, OVCAR3 and A2780 human ovarian cancer cell lines were purchased from American Type Culture Collection. OV75 primary ovarian cancer cells were provided by Dr Jocelyn Reader and Dr Dana Roque, who were the original suppliers (Division of Gynecologic Oncology, University of Maryland School of Medicine, Baltimore, MD, USA). OV75 cells are primary ovarian cancer cells available for research labs from the University of Maryland. The SKOV3, CAOV3 and A2780 cells were cultured in DMEM (Corning, Inc.) supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA) and 1% penicillin/streptomycin (PS). OVCAR3 cells were cultured in RPMI 1640 medium (Corning, Inc.) with 10% FBS and 1% PS. OV75 cells were cultured in HOSE Media [1:1 mixture of MCDB 105 (Sigma-Aldrich; Merck KGaA) and Medium 199 (Thermo Fisher Scientific, Inc.)], 10% FBS, 1% L-glutamine, 1% non-essential amino acids, 1% PS, sodium bicarbonate at pH 7.4) as described previously (17). All cell lines were grown in a humidified 37°C incubator with 5% CO₂/95% air, and the medium was replaced twice a week.

Immunoreactive IL-6 and IL-8 were measured using an Quantikine ELISA kit (Human CXCL8/IL-8, cat. no. D8000C; human IL-6, cat. no. D6050; R&D Systems, Inc.). Ovarian cancer cells were seeded in 6-well plates at a confluency of 70%. The cell-free culture supernatant of every well was collected and incubated, and optical density (OD) was read at 450 nm. Each assay detects cytokines as low as 5±7 pg/ml.

Cells were seeded in 10-cm plates at 70% confluency and treated with DMSO, Baze, SCH or Baze + SCH (CAOV3, Baze 10 µM, SCH 50 µM; SKOV3, Baze 5 µM, SCH 50 µM) at 37°C overnight before being harvested, lysed in cold Cell Lysis Buffer (0.5% 0.2 M PMSF, 0.5% 0.2 M NaF, 0.5% 0.2 M NaPP, 0.5% 0.1 M Na₃VO₄ and 4% 25X CP1; Cell Signaling Technology, Inc.) to collect the protein for western blot analysis. The protein concentration was determined using a Microplate BCA Protein Assay kit (cat. no. 23252; Thermo Fisher Scientific, Inc.). The proteins (30 µg/lane) were separated by 10% SDS-PAGE (30 µg per lane), transferred to a PVDF membrane at 350 mA for 110 min, blocked with 5% milk/TBS-8% Tween-20 (TBST) for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. The membranes were washed with TBST three times (15 min each), blotted with the secondary antibody for 1.5 h at room temperature. The visualization reagent (Western Lighting Plus-ECL; PerkinElmer, Inc.) was added and membranes were scanned using the Amersham Imager 600 (GE Healthcare, version 2.0.0).

The following primary antibodies were purchased from Cell Signaling Technology, Inc. and diluted at 1:1,000 in 5% milk: Phosphorylated (p)-STAT3 (Y705; rabbit mAb; cat. no. 9131S), STAT3 (rabbit mAb; cat. no. 4904S), p-S6 (rabbit mAb; cat. no. 4858S), S6 (rabbit mAb; cat. no. 2217S), p-AKT (rabbit mAb; cat. no. 4060S), AKT (rabbit mAb; cat. no. 4691S), survivin (rabbit mAb; cat. no. 2808S) and GAPDH (rabbit mAb; cat. no. 2118S). The secondary antibody was diluted at 1:10,000 in 5% milk (anti-rabbit IgG HRP-linked antibody; cat. no. 7074; Cell Signaling Technology, Inc.).

Ovarian cancer cells were seeded in 24-well plates at the same cell density, depending on the growth ability of each cell line (SKOV3, 2×10³ cells per well; CAOV3, 3×10³ cells per well; OVCAR3, 3×10³ cells per well), cultured overnight at 37°C and treated with DMSO, Baze, SCH or Baze + SCH at 37°C until the end of the assay (CAOV3, Baze 5 µM, SCH 15 µM; SKOV3, Baze 2.5 µM, SCH 15 µM; OVCAR3, Baze 2.5 µM, SCH 15 µM). The cell number of each well was then counted every 2 days after treatment (on days 2, 4, 6 and 8) to generate the growth curves. The cell density of cell growth assay is much lower than that of other assays, so the doses of Baze were less than those of other assays (CAOV3, Baze 5 µM; SKOV3, Baze 2.5 µM; OVCAR3, Baze 2.5 µM). However, the effects of different concentrations of SCH (such as 10 or 15 µM) were similar. Therefore, the SCH concentration remained constant in a number of experiments, including the MTT and wound healing/cell migration assays (the concentration of SCH here is 15 µM for each cell line).

A total of 3,000 cells in 100 µl medium per well were seeded into 96-well microtiter plates and treated overnight with DMSO, Baze, SCH or Baze + SCH (CAOV3, Baze 5 µM, SCH 15 µM; SKOV3, Baze 5 µM, SCH 15 µM; OVCAR3, Baze 5 µM, SCH 15 µM; OV75, Baze 10 µM, SCH 15 µM), followed by incubation at 37°C for 72 h. Each well was treated with 20 µl MTT, incubated for ~4 h and then combined with 150 µl N,N-dimethylformamide solution, followed by further incubation overnight at room temperature protected from the light. Cell viability was assessed by measuring absorbance at 595 nm for each well. The cell viability of DMSO control cells was set at 100% and the cell viability of drug-treated cells was compared with that of DMSO-treated cells. The combination index (CI) was then determined using CompuSyn software (www.combosyn.com; ComboSyn, Inc.). CI values of 1 indicate an additive effect, values of >1 an antagonistic effect and values of <1 a synergistic effect, based on the theorem of Chou (18).

SKOV3 cells were seeded in 6-well plates and incubated at 37°C overnight until they reached 100% confluence. A 100-µl pipette tip was used to scrape the monolayer of each well, each well was washed twice with PBS, serum-free culture medium containing different drugs (DMSO, Baze, SCH and their combination) was added (SKOV3, Baze 5 µM, SCH 15 µM) and images were captured under the light microscope.

Cells were incubated at 37°C and imaged again when the wound in the DMSO control was well healed (17 h). Migration inhibition was measured using ImageJ software 1.53e (National Institutes of Health) and calculated using the following formula: Percentage of wound healing = 100-[(final area/initial area) × 100%] (19).

This assay was performed as described previously (20). Matrigel was diluted at 1:8 with DMEM (without FBS and PS), added on the top of the Transwell membrane and allowed to polymerize for a minimum of 30 min at 37°C. Next, 5×10⁴ cells were added in 200 µl culture medium without FBS on the top of the Matrigel and 600 µl normal culture medium (DMEM or 1640 with 10% FBS) was added to the bottom of the lower chamber in a 24-well plate. The cells were treated with different drugs: DMSO, Baze, SCH or Baze + SCH for 24 h at 37°C (CAOV3, Baze 5 µM, SCH 15 µM; OVCAR3, Baze 10 µM, SCH 15 µM). The Transwell^® insert (permeable cell culture inserts; CELLTREAT Scientific Products) was then removed from the plate and the media and the remaining cells were carefully removed from the top of the membrane without damaging it using a cotton-tipped applicator. The Transwell insert was placed into 70% ethanol for 10 min and then stained with 0.2% crystal violet for 5-10 min at room temperature, rinsed, dried and imaged under a light microscope. Finally, the Transwell membrane was decolorized with 3% acetic acid to completely elute the crystal violet, and 100 µl/well eluent was added into a 96-well plate to measure the OD value (570 nm) on a microplate reader.

A peritoneal ovarian tumor mouse model was used to evaluate the efficacy of Baze + SCH compared with monotherapy in suppressing ovarian tumor growth. CAOV3-luciferase ovarian cancer cells (1×10⁷) in DMEM without FBS were injected intraperitoneally into a total of 20 female athymic nude mice aged 6–8 weeks purchased from Jackson Laboratory (21,22). Mice were housed at ~37°C with a 12/12-h light/dark cycle and ad libitum access to food and water. The development and growth of the tumors were monitored 1–2 times a week using an IVIS™ instrument (bioluminescent imaging; PerkinElmer, Inc.) (23). After the tumors had been measured, the mice were divided into four different groups with 5 mice/group: i) Vehicle, DMSO; ii) Baze, 8.8 mg/kg/mouse (24,25); iii) SCH, 25 mg/kg/mouse (16); and iv) Baze + SCH. Both drugs were administered daily by intraperitoneal injection. All mice were monitored using an IVIS instrument. Once experiments had been completed at 2 months after injection, tumors were harvested, weighed and snap-frozen in liquid nitrogen, and stored at −80°C. Tumor tissue was ground into powder, lysed and separated by SDS-PAGE to examine the expression of the downstream targets of IL-6 and IL-8 in different mouse groups, as described in the western blot analysis subsection. The method of euthanasia used for the mice was CO₂ asphyxiation followed by cervical dislocation (CO₂ was introduced into the chamber at a rate of 30-70% of the chamber volume per min to minimize distress). The maximum tumor diameter was 14.5 mm and the volume obtained was 988.2675 mm³. The use of mice was approved on June 11, 2019 by the Institutional Animal Care & Use Committee of the University of Maryland (Baltimore, MD, USA).

The development and growth of tumors were monitored by detecting the bioluminescence through an IVIS™ Imaging System, which has a high-sensitivity cooled charge-coupled camera mounted in a light-tight box (21,23). The images were collected and analyzed using Living Image™ software (Version:4.5.2.18424; PerkinElmer, Inc.). The mice were anaesthetized and injected intraperitoneally with d-luciferin potassium salt solution (150 mg/kg), and then placed into the imaging chamber. Bioluminescent images were collected per week (days 7, 14, 22, 29, 36, 44 and 62) throughout the duration of the study.

Mice were anesthetized using pharmaceutical grade isoflurane dispensed from a precision vaporizer in 100% O₂ delivery gas at 3.5–4.5% to an induction chamber with charcoal scavenger attached (scavenger was weighed before use and weight recorded on side of cannister to determine disposal point by weight gain). For Xenogen Imaging, there was a dedicated XGI8 gas inhalation anesthesia apparatus next to the Xenogen IVIS-200 imager. Once recumbent 20–30 sec and respiratory rate was noted to slow, isoflurane was stopped, the chamber was flushed with 100% O₂ (to reduce technician exposure to anesthetic when chamber is opened) and the animal was removed to continue anesthesia with isoflurane at 1.5–2.5% via face mask inside the imaging chamber. After the imaging was performed, mice were moved to a clean cage with bedding. Mice were monitored until able to ambulate normally prior to return to their assigned husbandry rooms.

The significance of associations was determined using GraphPad Prism 7 software (GraphPad Software, Inc.). Data for experiments performed in triplicate are presented as the mean ± standard error of the mean. One-way ANOVA and Tukey's post hoc test were used to analyze the statistical differences among groups. P<0.05 was considered to indicate a statistically significant difference.

The secretion of IL-6 and IL-8 was examined in human ovarian cancer cell lines. The present results demonstrated that OVCAR3, SKOV3 and CAOV3 human ovarian cancer cell lines, and OV75 primary human ovarian cancer cells, secreted both IL-6 and IL-8 at moderate to high levels [IL-6 (pg/ml): OVCAR3, 58.75±2.859; SKOV3, 191.4±7.469; OV75, 4,113±110.7; CAOV3, 12,517±11.15; IL-8 (pg/ml): OVCAR3, 112.4±6.138; SKOV3, 2,215±68.58; OV75, 18,757±233.3; CAOV3, 4,776±53.34; Fig. 1]. As a negative control, A2780 non-serous human ovarian endometroid adenocarcinoma cells did not secrete detectable IL-6 and IL-8. In general, endometrioid carcinoma is associated with an improved prognosis compared with serous ovarian cancer (26). These results supported the current hypothesis that IL-6 and IL-8 signaling pathways may serve as potential therapeutic targets for the most aggressive human ovarian cancer types.

IL-6 and IL-8 signaling can both regulate cell proliferation, adhesion, metastasis and invasion in human ovarian cancer cells (4,27–29). Therefore, cell proliferation may be decreased by IL-6 and IL-8 inhibitors (Baze and SCH). Thus, a cell proliferation assay was first performed. As shown in Fig. 2, the proliferation of the ovarian cancer cells was inhibited by each drug alone, but Baze + SCH treatment exerted a stronger inhibitory effect compared with each monotherapy.

It has been reported that IL-6 and IL-8 enhance the proliferation of ovarian cancer cells (6). Therefore, the cell viability of ovarian cancer cells may be inhibited by IL-6 and IL-8 inhibitors. MTT assays were performed using Baze + SCH in OVCAR3, SKOV3, CAOV3 and OV75 human ovarian cancer cells As shown in Fig. 3, Baze + SCH treatment exerted a stronger inhibitory effect on cell viability compared with each monotherapy. Furthermore, the CI of each of the four ovarian cancer cell lines was <1, indicating that the two drugs had a synergistic effect.

Cell migration and invasion are important steps in tumor metastasis, which can indicate a poor prognosis (30,31). IL-6 and IL-8 are necessary and sufficient to increase tumor cell migration (7). Therefore, cell migration and invasion may be reduced by the inhibitors of IL-6 and IL-8. A cell migration/wound healing assay was performed using the SKOV3 cell line, as the monolayer phenotypes of the other two cell lines were not suitable for this assay. An in vitro invasion assay was also performed in OVCAR3 and CAOV3 cells using Matrigel, to detect the inhibitory effect on cell invasion. Compared with the DMSO control, cell migration (Baze, 72.73%; SCH, 71.82%; Baze + SCH, 41.82%; DMSO vs. Baze/SCH, Baze + SCH vs. Baze/SCH, P<0.0001; Fig. 4A) and invasion (Fig. 4B and C) were inhibited by Baze and SCH monotherapy (CAOV3: Baze, 68.73%; DMSO vs. Baze, P<0.0001; SCH, 76.05%, DMSO vs. SCH, P=0.0002; Baze + SCH, 54.8%, Baze + SCH vs. Baze, P<0.01; Baze + SCH vs. SCH, P<0.001; OVCAR3: Baze, 70.3%; DMSO vs. Baze, P<0.001; SCH, 87.98%, DMSO vs. SCH, P<0.05; Baze + SCH, 53.06%, Baze + SCH vs. Baze, P<0.01; Baze + SCH vs. SCH, P<0.0001). Baze + SCH treatment resulted in a greater inhibitory effect on cell migration and invasion compared with each monotherapy. These results indicated that Baze + SCH may be used to treat or prevent ovarian cancer cell invasion.

Since there are several interactions between the IL-6 and IL-8 pathways (5,7), western blot analysis was performed to determine which downstream targeted genes were synergistically inhibited by these two drugs. Two ovarian cancer cell lines were seeded in 10-cm plates and treated with DMSO, a single drug or their combination. Protein expression levels were analyzed. Compared with those in cells treated with DMSO, the levels of p-STAT3, p-AKT, p-S6 and survivin were decreased by monotherapy but this was not significant in most cases (Fig. 5). The expression levels following the combination treatment were significantly lower than those of monotherapy and DMSO across all targets (p-STAT3/STAT3, p-AKT/AKT, p-S6/S6 and survivin/GAPDH). These results indicated that Baze + SCH synergistically inhibited the expression of downstream targeted genes (p-STAT3/STAT3, p-AKT/AKT, p-S6/S6 and survivin/GAPDH) of IL-6 and IL-8 pathways in human ovarian cancer cells, which may have effects on cell viability, migration and invasion.

The antitumor effect of Baze + SCH was investigated in vivo. CAOV3-luciferase ovarian cancer cells (1×10⁷) were injected intraperitoneally into female athymic nude mice. The mice were divided into four different groups, with 5 mice per group: i) Vehicle, DMSO; ii) Baze, 8.8 mg/kg; iii) SCH, 25 mg/kg; iv) Baze + SCH. As shown in Fig. 6A and B, a significant reduction in tumor growth was observed in the Baze or SCH single-treatment groups. However, combination treatment with Baze + SCH exhibited a greater inhibitory effect than monotherapy. In addition, compared with the DMSO control group, combination treatment could significantly reduce the levels of p-STAT3/STAT3, p-AKT/AKT, p-S6/S6 and survivin/GAPDH in the tumor tissues (Fig. 6C).

In conclusion, the present in vitro and in vivo findings indicated that the combined inhibition of IL-6 and IL-8 has the potential to suppress the growth of ovarian cancer and prevent tumor invasion.