In total, 3.01 mg nedaplatin from Selleck Chemicals (Houston, TX, USA) was dissolved in 1 ml dimethyl sulfoxide. ABT-737 was synthesized according to the literature (12), and its purity was determined to be >99% by high performance liquid chromatography. MG132 was purchased from Selleck Chemicals. All the following antibodies used for western blotting are rabbit anti-human: Anti-Mcl-1 polyclonal antibody (dilution, 1:500; catalog no., SC-819), anti-PARP polyclonal antibody (dilution, 1:500; catalog no., SC-7150) and anti-procaspase-3 polyclonal antibody (dilution, 1:500; catalog no., SC-7148) (Santa Cruz Biotechnology, Dallas, TX, USA); and anti-cleaved-caspase-3 monoclonal antibody (dilution, 1:1,000; catalog no., 9668; Cell Signaling Technology, Danvers, MA, USA). The mouse anti-human antibodies for western blotting are as follows: Anti-x-linked inhibition of apoptosis protein (XIAP) monoclonal antibody (dilution, 1:500; catalog no., SC-55550; Santa Cruz Biotechnology); and anti-β-actin monoclonal antibody (dilution, 1:2,000; catalog no., BD-612656; BD Biosciences, Franklin Lakes, NJ, USA).

Human lung cancer A549, NCI-H1299 and 95-D cell lines, prostate cancer PC-3 cell line and ovarian cancer SKOV3 cell line were obtained from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). All cell lines were tested and authenticated for genotypes by DNA fingerprinting. The 95-D, NCI-H1299 and SKOV3 cells were maintained in RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (HyClone; GE Healthcare Life Sciences, Logan, UT, USA), and the PC-3 and A549 cells were grown in Ham's F12 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum. All the cell lines were maintained in a humidified atmosphere of 95% air plus 5% CO ₂ at 37°C.

The anti-proliferative activity of nedaplatin plus ABT-737 was detected by SRB (Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) assay. Briefly, cancer cells were fixed with 10% trichoroacetic acid solution. Subsequent to washing, 0.4% SRB solution (100 µl per well) was added into each well. Following 20 min staining, wells were rinsed with 1% acetic acid to remove unbound dye, and then left to air dry. Subsequently, 100 µl Tris-base lye (10 mmol/l; Biosharp, Hefei, China) was added, followed by 10-min oscillation. The absorbance was then recorded at 515 nm using a multi-scan spectrum (Thermo Scientific Multiskan Go 1510; Thermo Fisher Scientific, Inc.).

Cancer cells were plated at 500–1,000 cells per dish, and then cells were exposed to nedaplatin (1 µM), ABT-737 (1 µM) or a combination of the two. After 14 days, dishes were stained by crystal violet and colony numbers were counted.

Sub-G1 analysis following PI staining was used to detect apoptosis. A549 cells (3×10⁵/well) were seeded into 6-well plates and exposed to either nedaplatin (20 µM) or ABT-737 (5 µM), or the two agents together. 95-D cells (3×10⁵/well) were seeded into 6-well plates and exposed to either nedaplatin (10 µM) or ABT-737 (10 µM), or the two agents together. Cells were harvested and washed with phosphate-buffered saline (PBS) three times and fixed with pre-cooled 70% ethanol at −20°C overnight. Cells were washed and resuspended in 500 µl PBS containing 50 µg/ml RNase at 37°C for 30 min. The cells were then stained with 5 µg PI at room temperature for 30 min. For each sample, 2×10⁴ cells were collected and analyzed using a FACS-Calibur cytometer (Becton Dickinson, San Jose, CA, USA), and the data were analyzed using Cellquest Software (version 6.0; Becton Dickinson, San Jose, CA, USA).

Cells (3×10⁵/well) were treated with nedaplatin and/or ABT-737 for 48 h, collected, and resuspended in fresh medium containing 10 µg/ml 5,5′,6,6′tetrachloro-1,1′,3,3′-tetraethylbenzimidazol-carbocyanine iodide (JC-1; Sigma-Aldrich; Merck Millipore). After incubation at 37°C for 30 min, cells were analyzed by FACS-Calibur cytometer.

Proteins were extracted with lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 0.1% sodium dodecyl sulfate, 1 mM ethylenediaminetetraacetic acid, 0.5% deoxycholic acid, 1% NP-40, 2.0 µg/ml aprotinin, 1 mM phenylmethylsulfonylfluoride and 0.02% sodium azide (Beyotime Institute of Biotechnology, Haimen, China). The lysates were centrifuged at 10,000 × g for 30 min at 4°C, then the concentrations protein were determined. Proteins were fractionated on 8–15% Tris-glycine gels, and then they were transferred to polyvinylidene fluoride membrane (Millipore, Bedford, MA, USA) and probed with the aforementioned primary antibodies (dilution range 1:500–1:1,000). The proteins were visualized with peroxidase-coupled secondary antibodies horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, catalog no., GAM007; HRP-conjugated goat anti-rabbit IgG, catalog no., GAR007; MultiSciences, Hangzhou, China) at a dilution of 1:5,000. Finally, proteins were visualized using the enhanced chemiluminescence detection system (PerkinElmer, Waltham, MA, USA).

RNA was isolated from A549 and 95-D cells using the TRIzol system (Thermo Fisher Scientific, Inc.), and the concentration of RNA was determined using NanoDrop 2000 (Thermo Fisher Scientific, Inc). Single-strand cDNA was prepared from the purified RNA using oligo (dT) priming (Thermoscript RT kit; Invitrogen; Thermo Fisher Scientific, Inc.), followed by SYBR-Green qPCR (Qiagen, Hilden, Germany). The sequences of PCR primers were as follows: Mcl-1 forward, 5′-GGGCAGGATTGTGACTCTCATT-3′ and reverse, 5′-GATGCAGCTTTCTTGGTTTATGG-3′; glyceraldehyde 3-phosphate dehydrogenase forward, 5′-GAGTCAACGGATTTGGTCGT-3′ and reverse, 5′-TTGATTTTGGAGGGATCTCG-3′ (Sangon Biotech, Shanghai, China).

The pTOPO-Mcl-1 plasmid from Addgene (15) (Plasmid 21605; Cambridge, MA, USA) or the empty vector (pTOPO) was transfected into A549 cells by Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol.

Two-tailed Student's t–test was used to detect the significance of differences between the experimental conditions. P<0.05 was considered to indicate a statistically significant difference. Combination index (CI) was used to quantify drug synergism, based on the multiple drug-effect equation of Chou-Talalay (16). For in vitro experiments, CI values were calculated for each concentration of nedaplatin, ABT-737 and the combination of the two in SRB assays using CalcuSyn (version 2.0; Biosoft, Cambridge, UK), and the mean CI values were presented.

The sensitivities of 5 human cancer cell lines to nedaplatin, ABT-737, or nedaplatin combined with ABT-737 were detected by SRB assay, and the survival curves are shown in Fig. 1. The fixed-ratio concentrations of nedaplatin and ABT-737 were used and CI values were calculated using CalcuSyn Software to assess combination activity (95-D, nedaplatin vs. ABT-737, 2:1; other cell lines, nedaplatin vs. ABT-737, 1:1). Nedaplatin plus ABT-737 showed synergistic effects in 5 human cancer cell lines, with the CI values <0.7. Nedaplatin at 1 µM and ABT-737 at 1 µM alone had limited effects on suppressing A549 cell colony formation; however, the combination almost eliminated colony formation in the colony formation assay (Fig. 2). Thus, combination was much more effective than either single agent in inhibiting the proliferation of cancer cells.

To investigate whether the cytotoxic effects of treatments were linked to the enhancement of apoptosis, PI staining was used to detect apoptosis in A549 and 95-D cells, which showed strong synergistic anti-cancer effects in cytotoxicity assay. As shown in Fig. 3A (top panel), the percentage of apoptotic A549 cells was markedly increased in the combination treatment group (66.4%) compared to ABT-737 (10.18%) or nedaplatin (19.81%) alone (ABT-737 vs. combination, P=0.004; nedaplatin vs. combination, P=0.005). Similarly, the apoptotic 95-D cells were significantly higher in the combination treatment group compared with the single treatment group (ABT-737 vs. combination, P=0.008; nedaplatin vs. combination, P=0.009; Fig. 3B and C).

To further assess whether increased ABT-737-mediated apoptosis by nedaplatin-induced caspase activation, immunoblot analysis was performed. As shown in Fig. 4, combined treatment markedly activated caspase-3 and PARP in both A549 and 95-D cells. In addition, combination treatment also resulted in potentiation of XIAP downregulation in A549 and 95-D cells. Overall, nedaplatin and ABT-737 showed a synergistic effect by inducing apoptosis in cancer cells.

Rapid loss of mitochondrial membrane potential and the release of cytochrome c are considered to be the hallmarks of mitochondrial dysfunction, which can induce the activation of caspases and lead to apoptosis (10). Thus, the present study investigated whether or not apoptosis induced by nedaplatin plus ABT-737 was triggered by mitochondrial dysfunction. As shown in Fig. 3A (bottom panel), nedaplatin plus ABT-737 resulted in an increased percentage of mitochondrial membrane depolarized A549 cells than compared with a single agent used alone (51.76% in combination treated cells, 23.04% in nedaplatin-treated cells, 18.00% in ABT-737-treated cells and 8.08% in control group; ABT-737 vs. combination, P=0.03; nedaplatin vs. combination, P=0.04). Combination treatment with nedaplatin and ABT-737 resulted in increased mitochondrial membrane potential in A549 and 95-D cell lines (Fig. 3D and E).

It has been reported that high levels of Mcl-1 may confer resistance to ABT-737 in several solid tumors (17). Thus, the present study examined the involvement of Mcl-1 in nedaplatin and ABT-737 combination treatment. Notably, it was found that treatment with nedaplatin or ABT-737 alone increased the expression of Mcl-1 in A549 and 95-D cells, whereas Mcl-1 expression was markedly decreased in the nedaplatin plus ABT-737 combination group, suggesting that Mcl-1 may be involved in the synergistic effect (Fig. 4). To determine whether the synergistic reduction of Mcl-1 protein by nedaplatin and ABT-737 combination treatment was the result of transcriptional inhibition, the level of Mcl-1 mRNA expression was assessed by RT-PCR in A549 and 95-D cells treated with ABT-737, nedaplatin, or a combination of the two for 12 h. As shown in Fig. 5A, no apparent synergist inhibitory effects on Mcl-1 mRNA expression levels were observed in the nedaplatin plus ABT-737 group in A549 and 95-D cells, whereas the Mcl-1 mRNA expression level was upregulated in nedaplatin plus ABT-737 combination-treated cells. Therefore, these data revealed that the synergistic reduction of Mcl-1 protein by nedaplatin and ABT-737 combination treatment was not the result of transcriptional inhibition. Thus, it was hypothesized that the putative ubiquitination of Mcl-1 in response to nedaplatin and ABT-737 combination treatment may play a key role in the synergistic effect. To further investigate this hypothesis, A549 cells were treated with CHX (200 mg/ml) to block new protein synthesis and observed Mcl-1 degradation in the presence of 5 µM ABT-737 and/or 20 µM nedaplatin. The half-life of Mcl-1 was compared in A549 cells treated with CHX in the presence of nedaplatin, ABT-737, or a combination of the two. Fig. 5B showed that the level of Mcl-1 protein decreased more rapidly in the nedaplatin plus ABT-737 group compared with either of the single-agent groups. Addition of the proteasome inhibitor MG132 attenuated combination-mediated Mcl-1 degradation (Fig. 5C). These data suggested that a promotion of Mcl-1 degradation was involved in the synergistic effect of nedaplatin and ABT-737.

To further evaluate whether the downregulation of Mcl-1 was required for nedaplatin plus ABT-737-induced apoptosis, the expression of Mcl-1 protein was successfully increased via transfecting pTOPO-Mcl-1 plasmid to A549 cells (Fig. 5D). Mcl-1 overexpression significantly decreased apoptosis in A549 cells treated with nedaplatin plus ABT-737 (Fig. 5E). The present results indicated that downregulation of Mcl-1 may contribute to the synergistic killing of cells by the combination of nedaplatin and ABT-737.