MDA-MB-435S, Caki, and A549 cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). Human skin fibroblasts (HSFs) were a gift from Dr T.J. Lee (Yeungnam University, Korea). The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 20 μM HEPES buffer, and 100 μg/ml gentamicin. ABT-737, VX-680 and MLN8237 were purchased from Sellek (Houston, TX, USA). Barasertib was purchased from Tocris (Ellisville, MO, USA). z-VAD was purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-death receptor 5 (DR5), anti-Bcl2, anti-Bcl-xL, anti-Mcl-1, anti-cIAP2 and anti-PARP antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-c-FLIP (L) antibody was obtained from Alexis Corporation (San Diego, CA, USA). The anti-actin antibody was obtained from Sigma-Aldrich.

The cells were suspended in 100 μl of phosphate-buffered saline (PBS), and 200 μl of 95% ethanol was added during a vortex step. The cells were incubated at 4°C for 1 h, washed with PBS and resuspended in 250 μl of 1.12% sodium citrate buffer (pH 8.4) together with 12.5 μg of RNase. The incubation was continued at 37°C for 30 min. The cellular DNA was then stained by applying 250 μl of propidium iodide (50 μg/ml) for 30 min at room temperature. The stained cells were analyzed by fluorescent-activated cell sorting on a FACScan flow cytometer for relative DNA content based on red fluorescence.

The cells were washed with cold PBS and lysed in ice in a modified RIPA buffer (50 μM Tris-HCl (pH 7.4), 1% NP-40, 0.25% Na-deoxycholate, 150 μM NaCl, 1 μM Na₃VO₄, and 1 μM NaF) containing protease inhibitors (100 μM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 10 μg/ml pepstatin and 2 μM EDTA). The lysates were centrifuged at 10,000 × g for 10 min at 4°C, and the supernatant fractions were collected. The proteins were separated by SDS-PAGE and transferred to an Immobilon-P membrane. The specific proteins were detected using an enhanced chemiluminescence (ECL) western blotting kit according to the manufacturer’s instructions.

A Cell Death Detection ELISA Plus kit (Boehringer Mannheim, USA) was used for assessing apoptotic activity by detecting fragmented DNA within the nucleus in the ABT-737-, VX-680-, and combined ABT-737 and VX-680-treated cells. Briefly, each culture plate was centrifuged for 10 min at 200 × g, the supernatant was removed, and the pellet was lysed for 30 min. After centrifuging the plate again at 200 × g for 10 min, the supernatant that contained the cytoplasmic histone-associated DNA fragments was collected and incubated with an immobilized anti-histone antibody. The reaction products were incubated with a peroxidase substrate for 5 min and measured by spectrophotometry at 405 and 490 nm (reference wavelength) with a microplate reader. The signals in the wells containing the substrate alone were subtracted as the background.

The possible synergistic effect of ABT-737 and VX-680 was evaluated using the isobologram method. Briefly, the cells were treated with different concentrations of ABT-737 and VX680 alone or in combination. After 48 h, relative survival was assessed and the concentration effect curves were used to determine the IC₅₀ (the half-maximal inhibitory concentration) values for each drug alone and in combination with a fixed concentration of the second agent (15).

To evaluate DEVDase activity, cell lysates were prepared after their respective treatments with ABT-737 in the presence or absence of VX-680. Assays were performed in 96-well microtiter plates by incubating 20 μg of cell lysates in 100 μl of reaction buffer (1% NP-40, 20 μM Tris-HCl, pH 7.5, 137 μM NaCl, 10% glycerol) containing a caspase substrate [Asp-Glu-Val-Asp-chromophore-p-nitroanilide (DVAD-pNA)] at 5 μM. Lysates were incubated at 37°C for 2 h. Thereafter, the absorbance at 405 nm was measured with a spectrophotometer.

Total RNA was isolated using TRIzol reagent (Life Technologies, Gaithersburg, MD, USA), and cDNA was prepared using M-MLV reverse transcriptase (Gibco-BRL, Gaithersburg, MD, USA) according to the manufacturer’s instructions. The following primers were used for the amplification of human Mcl-1 and actin: Mcl-1 forward, 5′-GCGACTGGCAAAGCT TGGCCTCAA-3′ and reverse, 5′-GTTACAGCTTGGATCC CAACTGCA-3′; Bcl-2 forward, 5′-GTCCTCAGCCCTCGC TCT-3′ and reverse, 5′-CACCTAATTGGGCTCCATCT-3′; c-FLIP forward, 5′-CCCAGTGGACAGCGAGC-3′ and reverse, 5′-ACTGCAGGCTTCCTGTGCGC-3′, actin forward, 5′-GGCATCGTCACCAACTGGGAC-3′ and reverse, 5′-CGA TTTCCCGCTCGGCCGTGG-3′. PCR amplification was carried out using the following cycling conditions: 94°C for 3 min followed by 17 (actin) or 23 cycles (Bcl-2, Mcl-1 and c-FLIP) of 94°C for 45 sec, 58°C for 45 sec, 72°C for 1 min, and a final extension at 72°C for 10 min. The amplified products were separated by electrophoresis on a 1.5% agarose gel and detected under UV light.

Transient transfection was performed in 6-well plates. One day before the transfection, MDA-MB-435S cells were plated at ~60–80% confluency. The Bcl-2/-3254 promoter-luciferase, NF-κB-luciferase, and cAMP response element (CRE)-luciferase plasmid were transfected into the cells using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA). The NF-κB-luciferase (plasmid no. 26699) plasmid was purchased from Addgene (Cambridge, MA, USA) (16). The CRE-luciferase plasmid was purchased from Clontech (Palo Alto, CA, USA). To assess the promoter-driven expression of the luciferase gene, the cells were collected and disrupted by sonication in lysis buffer (25 μM Tris-phosphate pH 7.8, 2 μM EDTA, 1% Triton X-100 and 10% glycerol), and aliquots of the supernatants were used to analyze the luciferase activity according to the manufacturer’s instructions (Promega, Madison, WI, USA).

The data were analyzed using a one-way ANOVA followed by post-hoc comparisons (Student-Newman-Keuls) using the statistical package for Social Sciences version 22.0 (SPSS Inc., Chicago, IL, USA).

Signaling molecule-target drugs inhibit specific signaling, which is different between cancer and normal cells, and a combination strategy reduces adverse effects, such as toxicity in normal cells. Therefore, we examined whether a combined treatment with sublethal dosages of ABT-737 and VX-680, which inhibits ATP-binding of Aurora kinase A, B and C (17), induces apoptosis in breast carcinoma MDA-MB-435S cells. ABT-737 plus VX-680 markedly induced cell shrinkage and membrane blebbing (Fig. 1A). To determine whether ABT-737 plus VX-680 induces apoptosis, FACS analysis to measure the DNA content and western blotting to detect the cleavage of PARP, a substrate of caspase-3, were performed. VX-680 induced G2/M arrest, but had no effect on apoptosis. However, the combined treatment with ABT-737 and VX-680 increased the sub-G1 population and PARP cleavage (Fig. 1B). In addition, ABT-737 plus VX-680 markedly increased cytoplasmic histone-associated DNA fragmentation (Fig. 1C). Next, to investigate whether the combined treatment with ABT-737 and VX-680 has a synergistic effect, MDA-MB-435S cells were treated with varied concentrations of ABT-737 and VX-680. The isobologram analysis revealed that the combined treatment with ABT-737 and VX-680 had synergistic effects (Fig. 1D). Next, to determine whether caspase is involved in the ABT-737 and VX-680-induced apoptosis, we examined caspase activity. ABT-737 and VX-680 increased caspase-3 activity, and a general caspase inhibitor, z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) markedly reduced apoptosis and PARP cleavage (Fig. 1E and F). These data revealed that the combined treatment with ABT-737 and VX-680 induced caspase-dependent apoptosis in the breast carcinoma MDA-MB-435S cells.

We investigated the effects of a combined treatment with ABT-737 and VX-680 on other types of cancer cells [human renal carcinoma (Caki cells), human lung carcinoma (A549 cells)] and normal cells (HSFs)]. Combined treatment with ABT-737 and VX-680 markedly induced apoptosis and PARP cleavage in the Caki and A549 cells (Fig. 2A and B). When MDA-MB-435S cells were treated with ABT-737 plus VX-680, the cell morphology was altered including cellular shrinkage and blebbing, while the morphology of the HSFs was not altered (Fig. 2C). Furthermore, combined treatment with ABT-737 and VX-680 had no effect on the sub-G1 population in the normal cells (Fig. 2C). Therefore, our results indicate that the combined treatment of ABT-737 and VX-680 induced apoptosis in cancer cells, but not in normal cells.

Next, we investigated the expression levels of the Bcl-2 family, the inhibitor of apoptosis (IAP) family and death receptors in ABT-737 and VX-680-treated cells. As shown in Fig. 3A, ABT-737 and VX-680 alone had no effect on apoptosis-related proteins, while the combined treatment with ABT-737 and VX-680 reduced c-FLIP, Bcl-2 and Mcl-1 protein expression. Furthermore, downregulation of Bcl-2 mRNA was detected, while the mRNA expression levels of c-FLIP and Mcl-2 were not altered in the ABT-737 plus VX-680-treated cells (Fig. 3B). To further confirm the mechanism of Bcl-2 downregulation in ABT-737 plus VX-680-treated cells, we examined whether the combined treatment with ABT-737 and VX-680 inhibits Bcl-2 promoter activity. As shown in Fig. 3C, ABT-737 plus VX-680 markedly reduced Bcl-2 promoter (Bcl-2/-3254) activity. Furthermore, we investigated whether the combined treatment with ABT-737 plus VX-680 inhibits NF-κB and CRE-associated transcriptional activities, which are important to Bcl-2 mRNA expression (18–20). NF-κB and CRE transcriptional activities were markedly reduced in the ABT-737 plus VX-680-treated cells (Fig. 3D). These results indicated that NF-κB and CRE transcriptional activities are involved in ABT-737 plus VX-680-mediated downregulation of Bcl-2 expression.

Combined treatment with ABT-737 and VX-680 downregulated Bcl-2, c-FLIP and Mcl-1 protein expression within 9, 18 and 24 h, respectively (Fig. 4A). To investigate whether downregulation of Bcl-2, c-FLIP and Mcl-1 is associated with ABT-737 plus VX-680-induced apoptosis, MDA-MB-435S cells were transiently transfected with Bcl-2, c-FLIP and Mcl-1. As shown in Fig. 4, overexpression of Bcl-2 or c-FLIP had no effect on the ABT-737 plus VX-680-mediated apoptosis, but overexpression of Mcl-1 partially blocked apoptosis in the ABT-737 plus VX-680-treated cells. These data revealed that combined treatment with ABT-737 and VX-680 induced apoptosis in the Bcl-2- or c-FLIP-overexpressed cells.

In the present study, we used VX-680, which inhibits both Aurora kinase A and B. To identify which Aurora kinase plays an important role in ABT-737-induced apoptosis, we tested more specific inhibitors of Aurora kinase A (MLN8237) and Aurora kinase B (barasertib). As shown in Fig. 5, both inhibitors of Aurora kinase A (MLN8237) and Aurora kinase B (barasertib) induced an increase in the sub-G1 population and PARP cleavage in the ABT-737-treated cells (Fig. 5A and B). Therefore, these results revealed that inhibition of both Aurora kinase A and B could be involved in the apoptosis in ABT-737-treated cells.