RIPA Lysis buffer was purchased from Beyotime Institute of Biotechnology. ABT-737 (a BH3 mimetic), N-Acety-L-Cysteine (NAC; ROS inhibitor), U0126 (ERK inhibitor), SB203580 (p38 inhibitor), SP600125 (JNK inhibitor), LY294002 (Akt inhibitor), GS-4997 (ASK1 inhibitor) and BAPTA-AM (calcium chelator) were purchased from Selleck Chemicals. Antibodies against Bcl-xL (cat. no. 551022; 1:200; mouse) and Bcl-2 (cat. no. 568664; 1:200; mouse) were purchased from BD Biosciences. Antibodies against Bax (cat. no. ab32503; 1:1,000; rabbit) and Bak (cat. no. ab32371; 1:1,000; rabbit) were purchased from Abcam. Antibodies against phosphorylated (p-) Ask1 (cat. no. 28846-1-AP; 1:1,000; rabbit), Ask1 (cat. no. 67072-1-Ig; 1:1,000; rabbit), p-P38 (cat. no. 28796-1-AP; 1:500; rabbit), P38 (cat. no. 14064-1-AP; 1:500; rabbit), p-Akt (cat. no. 66444-1-Ig; 1:2,000; mouse) and Akt (cat. no. 60203-2-Ig; 1:2,000; mouse) were purchased from Proteintech Group, Inc. Antibodies against caspase 3 (cat. no. 9662S; 1:2,000; rabbit), cleaved-caspase 3 (cat. no. 9661S; 1:1,000; rabbit), PARP (cat. no. 9542S; 1:1,000; rabbit), cleaved-PARP (cat. no. 9541S; 1:1,000; rabbit), p-JNK (cat. no. 4668S; 1:2,000; rabbit), JNK (cat. no. 9252S; 1:2,000; rabbit), p-ERK (cat. no. 4370S; 1:1,000; rabbit) and ERK (cat. no. 4695S; 1:1,000; rabbit) were purchased from Cell Signaling Technology, Inc. The antibody against β-actin (cat. no. AF0003; 1:2,000; mouse) was purchased from Beyotime Institute of Biotechnology.

Human cisplatin-resistant A2780/DDP cells were provided by the Department of Biochemistry and Molecular Biology (Basic Medical College, Shanxi Medical University). Cells were cultured in RPMI-1640 culture medium (Gibco; Thermo Fisher Scientific, Inc.) and supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. In total, 2 µg/ml cisplatin was used to maintain the A2780/DDP resistant line alive in cell culture.

Human cisplatin-resistant A2780/DDP cells were plated at 1.2×10⁴ cells/well in 96-well plates (Corning, Inc.). After incubating the cells for 24 h at 37°C with 5% CO₂, cells were treated as indicated. Next, 3-(4,5-dimethylthizaol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent [10 µl; 5 mg/ml in phosphate-buffered saline (PBS); Sigma-Aldrich; Merck KGaA] was added to each well, then the plates were incubated for 4–6 h. Formazan crystals were dissolved in 150 µl DMSO and the wells were shaken for 10 to 15 min using a tablet oscillation device. Absorbance values were measured at a wavelength of 490 nm (Bio-Rad Laboratories, Inc.). The mean value of five replicate wells was calculated for each treatment group.

Apoptosis was detected using a TUNEL (TdT-mediated dUTP Nick-End Labeling) In Situ Cell Death Detection Kit (Roche Diagnostics) according to the manufacturer's protocol. The principle of the TUNEL method is that apoptotic cells have DNA breaks in the genome, with the exposed 3′-OH groups able to be conjugated with fluorescein (FITC)-labeled dUTP catalyzed by terminal deoxynucleotidyl transferase (TdT). The apoptotic levels were detected using TUNEL assays by flow cytometry and confocal fluorescence microscopy. The detailed operation process was as follows:

When apoptosis levels were detected using TUNEL assays by flow cytometry, cells were harvested using trypsin (0.4%, Beijing Solarbio Science & Technology Co., Ltd.) and washed with PBS. Cells were fixed with 4% (w/v) paraformaldehyde (Beijing Solarbio Science & Technology Co., Ltd.)/PBS for 20 min on ice and washed with PBS. Cells were fixed with 70% (w/v) ethanol for 4 h at −20°C. The cells were centrifuged at 3,000 × g for 5 min at 4°C and washed with PBS, and then incubated with 0.1% Triton X-100 for 5 min at room temperature. After washing with PBS, the cells were incubated with proteinase K (20 µg/ml) for 8–10 min at room temperature, washed with PBS, and incubated with 80 µl equilibration buffer for 5 min at room temperature. The cells were centrifuged at 3,000 × g for 5 min at 4°C, then pellet was incubated with 50 µl terminal deoxynucleotidyl transferase (TdT) mixture (TdT: equilibration buffer, 1:9) for 1 h at 37°C in a humidified atmosphere. The cells were washed with PBS and resuspended in 200–300 µl PBS. Finally, the samples were examined by flow cytometry (Muse Cell Analyzer; MilliporeSigma). The results are representative of three independent experiments.

When apoptosis levels were detected using TUNEL assays by confocal fluorescence microscopy, A2780/DDP cells were plated at 4×10⁴ cells/well in 24-well plates (Corning, Inc.). After 24 h, the cells were treated as indicated at 37°C with 5% CO₂. The cells were washed with 0.1 M PBS three times and fixed with 4% (w/v) paraformaldehyde/PBS for 20–30 min at room temperature. After washing with 0.1 M PBS three times, the cells were incubated with 50 µl reaction solution mixture (TdT: FITC-12-dUTP labeling mix: equilibration buffer, 1:5:50) for 1 h at 37°C in a humidified atmosphere. The cell nuclei were stained by FITC-12-dUTP labeling mix, then apoptotic cells with characteristic nuclear fragmentation (green staining) were counted in six randomly chosen fields by confocal fluorescence microscopy (scale bar, 10 µm). The experiment was repeated three times.

The Hoechst staining method was used to examine apoptotic cells by staining the nuclei and observing cell morphological changes. A2780/DDP cells were cultured in 24-well plates (Corning, Inc.), then treated with the various indicated drugs for 24 h. Cells were washed with PBS three times and fixed with 4% (w/v) paraformaldehyde/PBS for 20–30 min at room temperature. After washing with PBS three times, the cells were incubated with Hoechst 33258/H₂O (2 µg/ml) for 5 min at room temperature, then washed with PBS three times. The cells were examined with a fluorescence microscope (Olympus Corporation). All samples were run in triplicate.

The Annexin V-FITC/PI Apoptosis Detection Kit (Dalian Meilun Biotechnology Co., Ltd.) was used to detect cell apoptosis levels at different stages based on the staining of Annexin V-FITC and PI. A2780/DDP cells were exposed to the indicated treatments for various times. The cells were harvested by trypsin (0.4%; Beijing Solarbio Science & Technology Co., Ltd.) and washed with PBS for two times. Then, 1X binding buffer was added to resuspend the cells to a concentration of 1×10⁶ cells/ml and 100 µl cell suspension (total of 1×10⁵ cells) was added into a new tube. Next, 5 µl annexin V-FITC and 5–10 µl PI were added, then the samples were gently mixed and incubated at room temperature in the dark for 15 min. After this staining incubation period, 400 µl 1X binding buffer was added to each tube, was mixed and detected by flow cytometry. The results were analyzed with FlowJo 10.8.1 software (BD Biosciences). A total of three replicates were performed for each sample.

A2780/DDP cells were cultured in 24-well plates. After incubation with the indicated treatments for 24 h, DCFH-DA (5 µM; Beyotime Institute of Biotechnology) was added to the cells. The cells were incubated at 37°C for 20 min, then washed three times with PBS to sufficiently remove any excess DCFH-DA. A fluorescence microscope (Olympus Corporation) was used to detect changes in intracellular ROS production in the A2780/DDP cells. The experiment was repeated three times.

A2780/DDP cells were lysed in 100 µl RIPA lysis buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) by sonication. Protein concentrations were quantified using a protein assay kit (Bio-Rad Laboratories, Inc.). Equivalent amounts of total proteins (30 to 40 µg) were separated using 12% SDS-poly-acrylamide gel electrophoresis and transferred onto PVDF membranes (MilliporeSigma). The membranes were blocked with 5% non-fat dry milk in PBST buffer (10 mM Tris-HCl pH 7.6, 100 mM NaCl and 0.1% Tween-20) for 90–120 min at room temperature, then incubated with the relevant primary antibodies overnight at 4°C. The membranes were washed with PBST buffer three times for 10 min each, then incubated with a horseradish peroxidase-conjugated goat anti-mouse secondary antibody (cat. no. A0216; Beyotime Institute of Biotechnology) or a horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (cat. no. A0208; Beyotime Institute of Biotechnology) at a 1:1,000 dilution for 90 min at room temperature. The membranes were washed with PBST buffer again three times for 10 min each. The immunoreactive bands were visualized by Enhanced Chemiluminescent (ECL) detecting agents (Thermo Fisher Scientific, Inc.) and measured with an ECL image detection system (BD Biosciences). The protein levels were quantified by densitometry using Quantity One 4.6.2 software (Bio-Rad Laboratories, Inc.). The data are presented as the mean ± standard deviation (SD) of three independent experiments.

A2780/DDP cells were cultured in 6-well plates and cultured at 37°C. After the cells were cultured to 80% confluence, they were incubated with indicated treatments for 24 h. The intracellular levels of hydrogen peroxide, superoxide anions, and hydroxyl radicals were detected using Hydrogen Peroxide assay kit (cat. no. S0038; Beyotime Institute of Biotechnology), Superoxide Anions assay kit (cat. no. BES-2343BTK; Shanghai Bolsen Biotechnology Co., Ltd.) and Hydroxyl Radicals assay kit (cat. no. BES20343BO; Shanghai Bolsen Biotechnology Co., Ltd.), respectively, according to the manufacturer's protocols. The data were measured using a microplate reader according to the manufacturer's protocols. A total of three replicates were performed for each sample.

A2780/DDP cells were cultured in 6-well plates at a density of 1×10⁵ cells/well, then incubated with the indicated treatments for 24 h at 4°C. Changes in the T-AOC were detected using the T-AOC Assay Kit (with ABTS method; cat. no. S0119; Beyotime Institute of Biotechnology) according to the manufacturer's protocol. A total of three replicates were performed for each sample.

Statistical analysis was performed using SPSS (version 22.0; IBM Corp.). The data are presented as the mean ± SD. One-way ANOVA was used for multiple-group comparisons, followed by Tukey's post hoc test. Differences between two groups were determined using an unpaired Student's t-test. P<0.05 was considered to indicate a statistically significant difference. Data are representative of three independent experiments performed in triplicate.

A2780/DDP cells were treated with different concentrations of ABT-737 (0, 2.5, 5, 10, 20, 40 and 80 µM) and cisplatin (0, 2.5, 5, 10, 20, 40 and 80 µg/ml) for 24 h or 48 h; then the cell survival rates were examined by MTT assays. The results showed that the cell viability decreased in a time- and dose-dependent manner (Fig. 1A and B). A literature review suggested that when examining the combined effect of the two drugs, it is best to choose the dose of each drug alone when the cell viability inhibition rate was ~10% (27). From the MTT assay results, 10 µM and 5 µg/ml were selected as the optimal concentrations of ABT-737 and cisplatin, respectively.

Next, to investigate if ABT-737 can affect the antitumor effects of cisplatin, A2780/DDP cells were treated with different concentrations of ABT-737 (10, 20, 40 µM) combined with cisplatin (5, 10 and 20 µg/ml) for 24 or 48 h, separately (Fig. 2A-C). From the aforementioned MTT assay results, the optimal treatment combination was selected: 10 µM ABT-737 combined with 5 µg/ml cisplatin for 24 or 48 h in A2780/DDP cells (Fig. 2D). The MTT assay results showed that ABT-737 enhanced cisplatin-induced proliferation inhibition in A2780/DDP cells.

It was then further investigated if ABT-737 could increase cisplatin-induced apoptosis in these cells. The data shown in Fig. 2D were analyzed and it was found that the cell survival rate was ~30% when cisplatin (5 µg/ml) and ABT-737 (10 µM) were combined for 24 h in A2780/DDP cells. Next, a literature research was performed and the optimal experiment condition of cells was considered, finding that 24 h was more suitable for subsequent experiments (27,28). Therefore, the cells were treated with cisplatin and/or ABT-737 for 24 h and the expression levels of apoptosis-related proteins Bcl-2, Bcl-xL, Bak, Bax, total-caspase 3, cleaved-caspase 3, total-PARP and cleaved-PARP were evaluated. The results revealed that the cisplatin and ABT-737 combination clearly increased the protein expression levels of Bax, Bak, cleaved-caspase 3 and cleaved-PARP, but decreased those of Bcl-2 and Bcl-xL (Fig. 3A-D). In addition, cells were treated with the cisplatin and ABT-737 combination for 24 h; then apoptotic levels were evaluated using TUNEL assays, flow cytometry and confocal microscopy, respectively. The result of the flow cytometry indicated that dUTP coupling fluorescence signal significantly increased in the cisplatin and ABT-737 combination group (Fig. 3E), and the data of the confocal microscopy demonstrated that there was increased DNA fragmentation in the cisplatin and ABT-737 combination group (Fig. 3F and G), preliminarily suggesting that ABT-737 could increase cisplatin-induced apoptosis. Based on these results, cells were treated with cisplatin and/or ABT-737 for 24 h and changes in nuclear morphology were examined using Hoechst 33258 staining. The results indicated that there were chromatin condensation and nuclear fragmentation in the cisplatin and ABT-737 combination group, suggesting that ABT-737 could increase cisplatin-induced DNA damage (Fig. 3H and I). To further confirm the apoptotic levels induced by cisplatin in combination with ABT-737, caspase 3 enzyme activity was detected using caspase 3 enzyme activity assay kit. The results showed a significant increase in caspase 3 enzyme activity in the cisplatin and ABT-737 combination group, suggesting that ABT-737 could increase cisplatin-induced caspase 3 enzyme activity (Fig. 3J). Taken together, these results suggested that ABT-737 treatment could increase cisplatin-induced apoptosis in A2780/DDP cells.

MAPK and PI3K/Akt are the main signaling pathways that regulate cell proliferation and apoptosis. It has been previously reported that ABT-737 can regulate different forms of apoptosis through the MAPK and PI3K/Akt signaling pathways (29). However, it is unclear whether ABT-737 can increase the sensitivity of ovarian cancer cells to cisplatin through these specific signaling pathways. Therefore, western blot analysis was used to determine the activation of MAPK and PI3K/Akt signaling pathways activation status in cells treated with ABT-737 and cisplatin combination for 24 h. The results demonstrated that the combination of ABT-737 and cisplatin could significantly increase the protein expression levels of p-JNK, p-ERK, p-p38 and p-Akt (Fig. 4A-E). In addition, specific inhibitors of the MAPK and PI3K/Akt signaling pathways were used, finding that 5 µM SP600125 (JNK inhibitor) treatment could significantly reverse the proliferation inhibition and apoptosis induced by the combination of ABT-737 and cisplatin. However, individual treatments with 5 µM U0126 (ERK1/2 inhibitor), 5 µM SB203580 (p38 inhibitor) and 5 µM LY294002 (Akt inhibitor) had no inhibitory effects on the slower proliferation and increased apoptosis induced by the ABT-737 and cisplatin combination (Fig. 4F-H). These data suggested that ABT-737 can increase the sensitivity of A2780/DDP to cisplatin through activation of the JNK pathway.

Although it was confirmed that ABT-737 could increase the sensitivity of A2780/DDP cells to cisplatin by activating the JNK signaling pathway, the upstream signaling details remained unclear. In numerous cell types, ASK1 has been found to play a vital role in activating the p38 and JNK signaling pathway (30). In the present study, western blot analysis was used to determine the activation of ASK1 protein induced by the combination of ABT-737 and cisplatin for 24 h. It was found that ABT-737 combined with cisplatin could effectively increase ASK1 protein phosphorylation levels (Fig. 5A and B). After treatment with the 5 µM ASK1 inhibitor GS-4997, MTT assays indicated that the inhibition of cell survival rate was significantly alleviated (Fig. 5C); Hoechst 33258 nuclear staining revealed that chromatin pyknosis and nuclear fragmentation were significantly reduced (Fig. 5D and E), and the result of Annexin V-FITC/PI double staining data demonstrated that cell apoptosis levels were significantly reduced (Fig. 5F and G). Collectively, these results suggested that ASK1 is important in ovarian cancer cells apoptosis induced by ABT-737. In addition, western blot analysis was used to detect the ASK1 and JNK protein phosphorylation levels after treatment with the ASK1 inhibitor GS-4997. These results showed that inhibiting ASK1 could significantly reduce JNK activation in these cells (Fig. 5H-J), indicating that ASK1 is necessary for ABT-737-induced JNK activation.

It has been previously reported that ABT-737 induces cell apoptosis by increasing oxidative stress, which is one of the main antitumor mechanisms of certain chemotherapeutic drugs. ROS play a vital role in maintaining the cell redox balance and can induce cell apoptosis (31). Firstly, ROS production in cells was detected after exposure to the ABT-737 and cisplatin combination for 24 h by fluorescence microscope. The results showed that ABT-737 combined with cisplatin could significantly increase ROS production in A2780/DDP cells (Fig. 6A and B). In addition, the levels of oxidative stress indices, including hydrogen peroxide, superoxide anions and hydroxyl radicals, were significantly increased following exposure to ABT-737 combined with cisplatin for 24 h (Fig. 6C-E), while the T-AOC levels of A2780/DDP cells were significantly reduced following combination treatment (Fig. 6F). These results indicated that ABT-737 could strongly enhance the imbalance between oxidation and antioxidant status induced by cisplatin in A2780/DDP cells.

Numerous studies have shown that oxidative stress can induce activation of the ASK1-JNK signaling pathway in numerous cell types (32,33). To further clarify the effects of ABT-737-induced oxidative stress on the ASK1-JNK signaling pathway, pretreatment NAC (an antioxidant) was first used to detect any ASK1 and JNK phosphorylation changes from the combined action of ABT-737 and cisplatin in A2780/DDP cells. The results identified that NAC could significantly inhibit the ASK1 and JNK protein phosphorylation levels (Fig. 7A-C), suggesting that the ROS formation induced by ABT-737 could mediate the activation of the ASK1-JNK signaling pathway. In addition, the effects of ABT-737-induced generation of ROS on cisplatin-induced apoptosis of A2780/DDP cells were detected. Following NAC treatment, MTT assays showed that the cell survival inhibition was significantly alleviated (Fig. 7D), while Hoechst 33258 nuclear staining indicated that chromatin pyknosis and nuclear fragmentation were significantly reduced (Fig. 7E and F). Furthermore, Annexin V-FITC/PI double staining demonstrated that the cell apoptotic rate was significantly reduced by NAC (Fig. 7G and H). These results identified that ROS production induced by ABT-737 could induce cisplatin-induced apoptosis in A2780/DDP cells. Overall, the data showed that ABT-737 treatment can induce ROS production in A2780/DDP cells, which mediates the activation of the ASK1-JNK signaling pathway and increases cisplatin-induced apoptosis. In summary, ABT-737 increases cisplatin sensitivity through the ROS-ASK1-JNK MAPK signaling axis in human ovarian cancer cisplatin-resistant A2780/DDP cells.