The stock solutions of ABT-737 were prepared in DMSO (Selleck Chemicals, Houston, TX, USA). Cisplatin, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG), Rotenone and 6-aminonicotinamide (6-AN) were from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). MitoSOX Red (mitochondrial super-oxide indicator) and MitoTracker Green were purchased from Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and antimycin A were purchased from Abcam (Cambridge, MA, USA). 2-deoxy-D-glucose (2-DG), rotenone and dehydroepiandrosterone (DHEA) were from Aladdin industrial Corporation (Shanghai, China). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). Anti-HK2 (cat. no. 22029-1-AP), anti-Bcl-2 (cat. no. 12789-1-AP), anti-G6PD (cat. no. 25413-1-AP), anti-β-actin (cat. no. 60008-1-Ig) and anti-PDHB (cat. no. 14744-1-AP) antibodies were purchased from ProteinTech Group, Inc., (Chicago, IL, USA) (1:1,000). Anti-HIF-1α (1:200; cat. no. sc-10790) and anti-Glut1 (1:200; cat. no. sc-7903) were purchased from Santa Cruz Biotechnology, Inc., (Dallas, TX, USA). Peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H+L; cat. no. SA00001-2) and peroxidase-conjugated AffiniPure goat anti-mouse IgG (H+L; cat. no. SA00001-1) from ProteinTech Group, Inc.

Human ovarian carcinoma cell lines SKOV3 (cisplatin-sensitive) and SKOV3/DDP (cisplatin-resistant) cells were obtained from the Chinese Academy of Medical Sciences and Peking Union Medical College (Beijing, China). The two cell lines were maintained in Roswell Park Memorial Institute-1640 (RPMI-1640) culture medium (Gibco Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS, Invitrogen; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin, and 100 mg/ml streptomycin (complete medium) at 37°C and 5% CO₂ with high humidity. To maintain resistance to cisplatin, SKOV3/DDP cells were cultured in complete medium with 1 μg/ml cisplatin (Sigma-Aldrich, Merck KGaA) (6).

Cell viability was determined by MTT assay. The cells were seeded in 96-well plates with 100 μl complete RPMI-1640 medium at 8×10³ per cells/well. Following overnight incubation at 37°C, increasing concentrations of cisplatin were applied followed by culture at 37°C for 24 h. For each group, replicate experiments were performed in five wells. A total of 20 μl MTT was added to each well and incubated for 4 h. Then, 150 μl DMSO was added to each well to dissolve the formazan crystals. The absorbance at 570 nm was determined using a microplate reader (BioTek Instruments, Inc., Winooski, VT, USA).

The expression levels of 84 key genes in glucose metabolism were determined by Human Glucose Metabolism RT² Profiler™ PCR Array (SABiosciences; Qiagen GmbH, Hilden, Germany). Total RNA was isolated from cultured cells, and 1 μg of total RNA was reverse-transcribed to single-stranded cDNA using the RT² First Strand kit (SABiosciences; Qiagen GmbH). The expression levels of genes of interest were determined by quantitative PCR using the Applied Biosystems 7300 Fast Real-Time PCR system (Thermo Fisher Scientific, Inc.) with SYBR Green fluorophore using the RT2 SYBR Green Master Mix (SABiosciences; Qiagen GmbH). The reaction program involved 40 cycles of 95°C for 10 min, 95°C for 15 sec and 60°C for 1 min. The results were analyzed using the manufacturer's software and relative gene expression was quantified using the 2^−ΔΔCq method (23). The altered expression of the 84 genes was displayed using heat imaging with normalization to β-actin.

In accordance with the manufacturer's protocol, total RNA of cultured cells was extracted using TRIzol (Invitrogen, Thermo Fisher Scientific Inc.). A total of 1 μg RNA was reverse-transcribed to cDNA using SuperScript™ IV First-Strand Synthesis System (Invitrogen, Thermo Fisher Scientific Inc.). The relative expression of genes of interest was determined with StepOne™ Real-Time PCR system and Power SYBR^® Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific Inc.) on an ABI 7300 instrument. The RT reaction was initially run at 50°C for 10 min, followed by a denaturation step at 94°C for 2 min. Also, the amplification reaction was continued for 35 cycles of denaturing (94°C for 15 sec) and annealing (55°C for 30 sec), followed by final extending step at 68°C for 1 min. The primer sequences are listed in Table I.

The rates of oxygen consumption (OCR) and extracellular acidification were determined using the fluorescent oxygen-sensitive and pH-sensitive probes Mito-Xpress and pH-Xtra (Luxcel Bioscience, Cork, Ireland). Briefly, SKOV3 or SKOV3/DDP cells were seeded in 96-well plates at 8×10⁴ cells/well. Following overnight incubation at 37°C, different drug treatments were applied followed by culture for 6 h. For each group, replicate experiments were performed in three wells (24).

The cells were seeded in 6-well plates at 5×10⁵ cells per well. Following overnight incubation at 37°C, the medium was changed to fresh complete medium. After 24 h, the medium of the cells was collected after which the proteins were extracted by sonication and quantified using the Bradford Protein Assay kit (Beyotime Institute of Biotechnology). Then, the glucose and lactate concentrations were determined using glucose assay (RsBio, Shanghai, China) and lactate assay kits (Jiancheng Bio, Nanjing, China), respectively. The untreated groups were collected and measured using 2-NBDG (50 μM; Sigma-Aldrich; Merck KGaA) in Dulbecco's modified Eagle's medium (Gibco Life Technologies; without serum and glucose) for 30 min to determine the capacity for glucose uptake using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

A total of 1×10⁶ cells were plated in a 25 cm²-cell culture flask. Following overnight incubation at 37°C, the medium was changed to fresh medium. After 24 h, in accordance with the manufacturer's instructions, cells were washed with PBS and then their ATP levels were determined by CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Madison, MI, USA). The glycogen concentrations were determined from the cell lysate using a glycogen assay kit (Jiancheng Bio) in accordance with the manufacturer's protocol.

Western blot determination of cell extracts was measured as described previously (25). After different treatments, the SKOV3 or SKOV3/DDP cells were harvested and washed with cold phosphate-buffered saline (PBS), and then incubated in ice-cold radioimmunoprecipitation assay buffer. The cell lysates were sonicated and centrifuged at 5,000 × g for 10 min. The concentration of the protein was determined using the Bradford Protein Assay kit (Beyotime Institute of Biotechnology). The proteins samples (30-50 μg) were separated by 12% w/v SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes. Then, the membranes were blocked with 5% (w/v) skim milk in PBST buffer [100 mM NaCl, 10 mM Tris-HCl (pH 7.6) and 0.1% (v/v) Tween-20] for 1 h at room temperature, and incubated overnight at 4°C with the primary antibody (anti-HK2, 1:1,000 dilution; anti-Bcl-2, 1:1,000 dilution; anti-G6PD, 1:1,000 dilution; anti-β-actin, 1:1,000 dilution; anti-PDHB, 1:1,000 dilution; anti-HIF-1α, 1:200 dilution and anti-Glut1, 1:200 dilution). The following day, the membranes were washed with PBST and incubated with secondary antibodies (1:2,000 dilution). The membranes were detected using the ECL reagents and captured using the Syngene Bio Imager (Synoptics Ltd., Cambridge, UK).

A total of 1×10⁶ SKOV3 or SKOV3/DDP cells were seeded in a 25 cm²-cell culture flask. Following overnight incubation at 37°C, the complete medium was changed to fresh medium. After 24 h, 5×10⁶ cells were collected followed by protein extraction by sonication and quantification using the Bradford Protein Assay kit. Then, the cells were subjected to analysis with the NADP⁺/NADPH Quantification kit (BioVision, Inc., Milpitas, CA, USA) to determine NADPH levels and NADPH/NADP⁺ ratios. The cellular glutathione (GSH) and glutathione disulfide (GSSG) levels and GSH/GSSG ratio were determined using commercial colorimetric kits (Jiancheng Bio) (26). The glucose 6-phosphate dehydrogenase (G6PD) activity was determined using commercial colorimetric kits (BioVision Inc.). The absorbance was determined using a microplate reader (BioTek Instruments, Inc.).

A total of 5×10⁵ cells were plated in 6-well plates and incubated overnight at 37°C. Following exposure to ABT737 and/or cisplatin at 37°C for 24 h, the cells were collected. The induction of apoptosis in these cells was determined using Annexin V-FITC (Annexin V Apoptosis Detection Kit II, BD Biosciences, San Diego, CA, USA). The cells were analyzed using a flow cytometer (BD Biosciences).

The intracellular reactive oxygen species (ROS) and intramitochondrial superoxide anion (O₂⁻) were also determined using DCFH-DA and MitoSOX Red, respectively. A total of 5×10⁵ cells were plated in 6-well plates and incubated overnight at 37°C. Following treatment as indicated in the figure legends, the cells were washed with PBS. DCFH-DA (10 μM) or MitoSOX Red (5 μM) was added to the cells and cultured at 37°C and 5% CO₂ for 20 min. Then, the cells were collected. The fluorescence of the stained cells was detected using a flow cytometer (BD Biosciences).

Cell death was detected using Calcein-AM/PI Double Stain Kit (Shanghai Yeasen Biotechnology Co., Ltd, Shanghai, China). Briefly, following treatment, the SKOV3 or SKOV3/DDP cells were cultured with 2 μM propidium iodide and 4 μM calcein acetoxymethyl ester in an incubator at 37°C and 5% CO₂ for 30 min. After rinsing with PBS, the viability of the cells was then determined using an IX71 fluorescence microscope. The dead and alive cells represented by bright red or green fluorescence were identified upon excitation at 544 and 485 nm, respectively.

The data were analyzed by one-way analysis of variance using SPSS (version 21.0; IBM Corp., Armonk, NY, USA). Tukey's post hoc test was used to determine the significance for all pairwise comparisons of interest. The data were obtained from three experiments and presented as the mean ± standard deviation. P<0.05 was considered to indicate a statistically significant difference.

A pair of isogenic ovarian cancer cell lines that were cisplatin-sensitive (SKOV3) or cisplatin-resistant (SKOV3/DDP) was used. SKOV3/DDP, a subline of SKOV3, acquired resistance to cisplatin in vitro (5). As expected, SKOV3/DDP cells exhibited considerable resistance to cisplatin, while SKOV3 cells also exhibited resistance to cisplatin as determined by the MTT assay following exposure to increasing concentrations of cisplatin for 24 h (Fig. 1A). As shown in Fig. 1B, SKOV3/DDP cells were preferentially enriched for G0/G1 quiescent cells and had a lower proliferation rate. The expression of genes associated with glucose metabolism was assessed by RT2 Human Glucose Metabolism Profiler PCR array. The obtained results indicated the upregulation of glycolysis, the tricarboxylic acid cycle (TCA) cycle and gluconeogenesis in SKOV3/DDP cells (Fig. 1C and Table II).

Further study suggested that glucose metabolism in SKOV3/DDP cells may be altered compared with SKOV3 cells with increased glucose uptake and consumption (Fig. 2A and B), decreased lactate production (Fig. 2C), and overexpression of the glucose metabolism-associated genes, PFKL and LDHA, and the glucose transporter Glut1 as well as elevated glycogen levels (Fig. 2D). As glycogen is a branched polymer of glucose that acts as an intracellular glucose store, high glycogen levels may render the cells less sensitive to glucose deprivation (Fig. 2E). Notably, SKOV3/DDP cells exhibited reduced sensitivity to glucose deprivation compared with SKOV3 cells (Fig. 2F), while the combined treatment with 2-DG (glycolysis inhibitor) induced significant cell death compared with the glucose deprivation alone group (Fig. 2G).

Numerous studies have previously demonstrated that the Warburg effect is extremely important to ovarian tumor growth (27,28). An analysis of the extracellular acidification rate (ECAR) indicated that ECAR was significantly lower (Fig. 3A) in SKOV3/DDP cells compared with SKOV3 cells, indicating a reduction of the Warburg effect in cisplatin-resistant cancer cells. 2-DG, a glycolytic inhibitor, blocks glycolysis by inhibiting hexokinase, which is the key rate-limiting enzyme of glycolysis. The results of the present study suggested that SKOV3/DDP cells were less sensitive to 2-DG compared with SKOV3 cells (Fig. 3B). As the basal rate of glycolysis in SKOV3/DDP cells was lower compared with SKOV3 cells, the metabolic status of SKOV3/DDP cells might involve downregulation of glycolysis and a shift toward OXPHOS. To investigate this hypothesis, the OCR (which is indicative of OXPHOS) in SKOV3 and SKOV3/DDP cells was measured. As shown in Fig. 3C, SKOV3/DDP cells demonstrated significantly higher OCR, which represented higher oxidative metabolism compared with SKOV3 cells. Consistent with this finding, the intracellular oxygen concentration was lower in SKOV3/DDP cells compared with SKOV3 cells (Fig. 3D). Furthermore, the ATP level was higher in SKOV3/DDP compared with SKOV3 cells (Fig. 3E).

As oxidative processes mainly take place in the mitochondria, whether the increased oxygen consumption demonstrated in SKOV3/DDP cells was a result of increased mitochondrial mass was investigated.

Notably, compared with the level in SKOV3 cells, SKOV3/DDP cells exhibited a marked increase in staining with MTG (MitoTracker green) (Fig. 4A). Furthermore, SKOV3/DDP cells exhibited a marked increase in labeling with the mitochondrial-specific redox probe, MitoSox-Red, suggesting that increased ROS content originated from the mitochondria and that mitochondria were the main source of oxidative metabolism in SKOV3/DDP cells (Fig. 4B). Moreover, the level of intracellular ROS in SKOV3/DDP cells was markedly increased in comparison to SKOV3 cells (Fig. 4C). Given their increased oxygen consumption, SKOV3/DDP cells exhibit a more pro-oxidant state.

To investigate whether the inhibition of mitochondrial respiration was able to lead to oxidative stress and induce the death of SKOV3/DDP cells, the complex I inhibitor, rotenone, was used. It was found that the intracellular levels of ROS that were detected by fluorescence of the redox dye DCFH-DA, decreased (Fig. 4D). By contrast, the number of dead cells, as detected by bright red fluorescence in the live/dead cell viability assay, was markedly increased upon treatment with cisplatin plus rotenone compared with treatment with cisplatin alone (Fig. 4F). Therefore, it was hypothesized that the marked cell death induced by rotenone may be associated with energy depletion. It was found that the treatment with rotenone and/or cisplatin caused a marked reduction in the ATP level in SKOV3/DDP cells (Fig. 4E). Notably, the present study demonstrated that compared with SKOV3 cells, SKOV3/DDP cells might rely more on the respiration of mitochondria rather than the Warburg effect to meet their energy demands.

The level of the antioxidant molecule, NADPH, which can be used to scavenge ROS (29), increased in SKOV3/DDP cells compared with SKOV3 cells (Fig. 5A). The ratio of NADPH to NADP⁺ in SKOV3/DDP cells also increased relative to the ratio in SKOV3 cells (Fig. 5B). GSH, an important redox buffer in cancer cells, has been considered to participate in sustaining cisplatin resistance (30). In SKOV3/DDP cells, GSH and GSSG contents, total GSH (GSH plus GSSG), and the GSH to GSSG ratio were significantly higher than those in SKOV3 cells (Fig. 5C–F). Moreover, the oxidative PPP branch is a major source of NADPH for cells, and substantial evidence has demonstrated that cancer cells mainly rely on the oxidative PPP branch to maintain redox homeostasis (31). Therefore, the authors hypothesized that SKOV3/DDP cells might exploit the PPP pathway to increase GSH biosynthesis to compensate for the increased ROS generated by OXPHOS. G6PD is a major rate-limiting enzyme for the activity of PPP. Notably, the gene and protein expression of G6PD (Fig. 5G) as well as its activity (Fig. 5H) were increased in SKOV3/DDP cells compared with the levels in SKOV3 cells.

To better understand the role of PPP in cisplatin resistance, the cells were treated with the competitive G6PD inhibitor 6-AN (6-aminonicotinamide) (32) or the uncompetitive G6PD inhibitor DHEA (dehydroepiandrosterone) (33) with or without 6 μg/ml cisplatin. In SKOV3 cells, the treatment of 6-AN or DHEA together with cisplatin for 24 h induced no significant effect on cell viability compared with the cisplatin alone group (Fig. 5I). By contrast, in SKOV3/DDP cells, treatment with 6-AN or DHEA together with cisplatin significantly reduced cell viability compared with the cisplatin alone group (Fig. 5I). These results suggested that SKOV3/DDP cells exploit oxidative PPP as a resistance mechanism. These results also demonstrated that SKOV3/DDP cells utilize the oxidative PPP branch as a mechanism of resistance to cisplatin by contributing to redox buffering.

SKOV3/DDP cells exhibit an overexpression of the Bcl-2 protein, which might render them more resistant to cisplatin (Fig. 6A and B). The increased expression of Bcl-2 in SKOV3/DDP cells is potentially important because Bcl-2 not only participates in the apoptotic pathway but also is involved in mitochondrial metabolism (34). To examine the mechanisms of Bcl-2 in cisplatin resistance, the cell survival rate was examined via MTT assays following exposure to various doses of the Bcl-2 inhibitor, ABT737, for 24 h. SKOV3/DDP cells were more sensitive to ABT737 than SKOV3 cells. Based on these MTT results, we treated both cell lines with 10 μM ABT737 (Fig. 6C). The effects of ABT737 on glucose metabolism-associated genes and OCR were analyzed in the two cell lines to clarify whether ABT737 affects glucose metabolism before exhibiting notable cytotoxicity, such as by inducing apoptosis. Treatment of the cells ABT737 with or without cisplatin was able to induce a significant decrease in the expression of glucose metabolism-associated genes and a decrease in OCR in SKOV3/DDP cells compared with the control group (Fig. 6D and E), but it had no marked effect on either the expression of glucose metabolism-associated genes or OCR in SKOV3 cells. The ATP level was significantly decreased in SKOV3/DDP cells upon treatment with cisplatin plus ABT737 compared with single treatment of cisplatin or ABT737 (Fig. 6F). Annexin V-FITC staining indicated that treatment with cisplatin plus ABT737 induced significant apoptosis in SKOV3/DDP cells compared with other treatment groups (Fig. 6G and H). These findings indicated that ABT737 induced significant cytotoxicity mainly by inhibiting the respiration of mitochondria in SKOV3/DDP cells and sensitizing the cells to cisplatin.

Notably, the expression of HIF-1α was inhibited by ABT737 (Fig. 7A–C), suggesting that ABT737 had a direct effect on HIF-1α that in turn may affect glycolysis. Since ABT737 could reduce the respiration of mitochondria in SKOV3/DDP cells, we proposed that the combined treatment of ABT737 and glycolysis inhibitor 2-DG would play an important therapeutic role in SKOV3/DDP cells. To estimate the effect of ABT737 and 2-DG in combination, we examined changes in the expression of enzymes associated with glucose metabolism (Fig. 7D). In SKOV3/DDP cells, among several proteins related to glucose metabolism, Bcl-2, HIF-1α, Glut1, HK2, G6PD, IDH1 and PDHB exhibited marked decreases in expression following this combination treatment compared with those in the other groups. Compared with that in SKOV3 cells, the GSH level in SKOV3/DDP cells was markedly decreased following treatment with ABT737 plus 2-DG compared with that following treatment with either ABT737 alone or 2-DG alone (Fig. 7E), while the intracellular ROS level in SKOV3/DDP cells was increased accordingly (Fig. 7F). Furthermore, we observed a remarkable increase of the death (Fig. 7G) of SKOV3/DDP cells treated with ABT737 and 2-DG compared with that in the other groups. These results indicate that the glycolysis inhibitor 2-DG enhanced the activation of apoptosis induced by ABT737.