Inhibition of mitotic kinase Aurora suppresses Akt-1 activation and induces apoptotic cell death in all-trans retinoid acid-resistant acute promyelocytic leukemia cells

VX-680 (Kava Tech, CA) was dissolved in dimethlsulfoxide (DMSO) to a stock concentration of 430 μM and stored at -20°C. Human APL NB4 and NB4-R2 cell lines, provided by Shanghai Institute of Hematology, Ruijin Hospital, were cultured in RPMI 1640 (Gibco) supplemented with 10% fetal bovine serum (FBS; Hyclone) at 37°C in a humidified 5% CO₂ atmosphere.

To measure CD11b expression, NB4 and NB4-R2 cells (5 × 10⁵/ml) were plated in 6-well dishes and cultured with ATRA (1 μM). After 3 days, Cells were washed twice with PBS and incubated with primary mouse monoclonal CD11b antibody (Sigma) at 37°C for 1 hr. Then, the cells were washed once with PBS, and incubated with the secondary immunofluorescence antibody (FITC) for 1 hr in dark. Expression of CD11b on cell surface was measured by flow cytometry.

NB4-R2 cells were incubated with VX-680 at 2 nM for 24 hr. Cells were fixed in cold methanol for 20 min at 4°C and permeabilized in 0.5% TritonX-100 in PBS at room temperature (RT) for 15 min. Then cells were incubated with 1% BSA for 1 hr at RT to block nonspecific binding before the primary antibody reaction. Slides were incubated with the primary antibody to Aur-A, α-Tubulin at RT for 1 hr, followed by Alexa Flour 680 or FITC 488 conjugated antibody. After counterstained with DAPI (1 μg/ml), cells were visualized using a microscope (1000 ×, Olympus).

Cell proliferation was assessed by MTT assay. NB4-R2 cells were plated in 96-well plates at 2.5 × 10⁴ cells/ml in a final volume of 200 μl and exposed to different doses of VX-680 (0-10 nM) or ATRA. Sets of 5-wells were used for each dose. 20 μl of MTT solution (Sigma, 5 mg/ml) was added to each well at 24 hr and 48 hr. After cells were incubated at 37°C for another 4 hr, the medium was removed and 150 μl DMSO was added to solubilize the formazan. Finally, the absorbance (OD) was measured using a multiwell plate reader (Bio-Rad Microplate Reader).

NB4-R2 cells were collected and washed twice with PBS, then fixed by ice alcohol overnight at -20°C. Cells were then resuspended with PI at a concentration of 1.0 × 10⁶ cells/ml. Quantification of Sub G1 population after PI staining was carried out using a FACS flow cytometer equipped with CellQuest software (BD).

After collecting and washing twice with PBS, VX-680 treated or untreated NB4-R2 cells were resuspended in the binding buffer (500 μl). FITC-Annexin-V (5 μl) was added to the cells followed by addition of 5 μl PI according to the protocol of the Annexin V-FITC/PI kit (EMD Biosciences). The samples were then incubated for 15 min in the dark at 4°C and subjected to flow cytometry evaluation.

Nuclear morphology of control and VX-680 treated cells was observed by staining cell nuclei with Hoechst 33342 (Sigma). Cells (at least 200 per slide) were incubated with Hoechst 33342 (10 μg/ml) for 15 min at RT and examined under a fluorescence microscope (Olympus) by using the MNU2 filter. Apoptotic cells were characterized by condensation of chromatin and/or nuclear fragmentation.

JC-1 probe was employed to measure mitochondrial depolarization in NB4-R2 cells. Briefly, VX-680 treated cells were incubated with an equal volume of staining solution (5 μg/ml) at 37°C for 20 min and rinsed twice with PBS. Mitochondrial membrane potentials were monitored by determining the relative amounts of dual emissions from mitochondrial JC-1 by flow cytometry. Mitochondrial depolarization was indicated by an increase in the green fluorescence and a decrease in the red fluorescence intensity.

NB4-R2 cells were lysed in RIPA buffer. The protein concentration was determined by Bradford method with BSA (Sigma) as the standard. Equal amounts of cell extract (40 μg) were subjected to electrophoresis in SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Minipore). The membrane was blocked and then incubated with GAPDH (from Ambion), p-Aur-A/AIK (Thr288), cleaved PARP (Asp214), pAkt-1 (Ser473), cleaved caspase-3 (Asp175) and pGSK-3 (Ser9) antibodies (from Cell Signaling), at 4°C overnight, followed by incubation for 1 hr RT with appropriate secondary antibodies. Antibody binding was detected with an enhanced chemiluminescence kit and ECL film.

Statistical analysis was performed using SPSS version 11.0 (SPSS Inc.). The Student's t-test was used to make a statistical comparison between groups. The level of significance was set at p < 0.05.

In order to demonstrate the specificity of Aurora inhibitory VX-680 on leukemia, OCI-AML3, NB4, HL-60 and ML-1 cells were treated with different doses of VX-680. As showed in Figure 1, VX-680 could inhibit cell growth rates in the 4 different leukemic cells we tested in a dose-dependent manner (ranging from 1 nM to 10 nM) after 24 hr treatment. However, VX-680 suppressed the proliferation in some solid tumor cell types with less potency, such as MCF-7 and Hela cancer cells (Figure S1, Additional file 1), suggesting that VX-680 was a potential anti-leukemic agent for various leukemic cell types.

Promyeloid leukemic cell lines NB4 and NB4-R2 were treated with ATRA and cell differentiation was evaluated by quantifying CD11b expression, a marker of myeloid differentiation. After exposure of NB4 and NB4-R2 cells to ATRA (1 μM) for 72 hr, a mean of 10.76% NB4 cells were induced to express cell surface antigen CD11b. On contrast, only 1.4% of NB4-R2 cells expressed CD11b surface antigen (Figure 2A, B), confirming that NB4-R2 cells were resistant to ATRA-induced myeloid differentiation. MTT assay further showed that ATRA (1 μM) significantly inhibited NB4 cells growth, while the survival percentage was not statistically changed at this concentration in NB4-R2 cells (Figure 2C), indicating ATRA failed to inhibit NB4-R2 cells growth.

We studied the inhibition of Aurora kinases in NB4-R2 cells using VX-680. Aur-A activation was inhibited by VX-680 at different concentrations (1 nM, 2 nM, 5 nM, 10 nM) in a dose-dependent manner in NB4-R2 cells (Figure 3A). VX-680 (5 nM) significantly inhibited Aur-A by reducing autophosphorylation at the activation site, Thr288. Then, we examined the role of Aur-A inhibition by VX-680 in the formation of spindles. As assessed by immunofluorescence, control cells displayed normal bipolar spindles, presenting a clearly visible metaphase plate straddled by uniform radial arrays of microtubules from opposite poles (Figure 3B). In the contrast, VX-680 (2 nM) treated cells showed abnormal monopolar spindles, suggesting that the inhibition of Aurora kinase activity induced defects of mitotic spindle in VX-680 treated cells.

Next, we studied if VX-680 could suppress proliferation in NB4-R2 cells in vitro. NB4-R2 cells were treated with VX-680 at the concentration of 1 nM, 2 nM, 5 nM and 10 nM for 24 hr and 48 hr. Cell viability was assessed by MTT assay. At the concentration of 5 nM and 10 nM, VX-680 significantly inhibited the growth of NB4-R2 cells, with IC50 value of the anti-proliferation effect of VX-680 at 7.10 nM for 24 hr and 4.29 nM for 48 hr in NB4-R2 cells (Figure 4A).

We further assessed whether VX-680 could induce apoptosis in NB4-R2 cells. Incubation of VX-680 (1 nM, 2 nM, 5 nM and 10 nM) led to an increased apoptosis for 24 hr (7.3%, 10.45%, 31.9% and 48.27%, respectively) and 48 hr (9.77%, 16.83%, 43.8% and 67.85%, respectively) by assessing the sub-G1 population (Figure 4B). In addition, apoptotic cells were also detected by both Annexin V/PI staining and immunofluorescent staining with Hoechst 33342. Annexin V/PI staining showed that percentage of apoptosis were 3.66%, 5.52%, 15.83%, 24.43% respectively for 24 hr, and 4.35%, 7.47%, 32.77%, 90.4% respectively for 48 hr at the indicated doses of VX-680 (Figure 5). Similarly, control cells which were stained by Hoechst 33342 were uniformly blue in viable cells, whereas the apoptotic cells showed bright blue dots in the nuclei, representing the nuclear fragmentation, especially at VX-680 concentration of 5 nM and 10 nM (Figure 6). These results indicated that the apoptotic NB4-R2 cells were induced by Aurora kinase small-molecule inhibitor VX-680 in both dose- and time-dependent manners.

Further, we investigated the molecule events triggered by Aurora inhibition. Reduction of mitochondrial membrane potential is one of the molecule events for early apoptosis. Changes in mitochondrial membrane potential was assessed by monitoring JC-1, which accumulates in mitochondria forming red fluorescent aggregates at high membrane potential, whereas exits mainly in cytosol forming green fluorescent monomer, presenting a collapse of membrane. In our study, VX-680 treated cells showed loss of red fluorescence and production of obvious green fluorescence, suggesting reduction of mitochondrial membrane potentials. At different concentrations of VX-680 (1 nM, 2 nM, 5 nM and 10 nM), the percentage of NB4-R2 cells emitted green fluorescence was 20.9%, 21.8%, 48.5% and 91.7%, respectively, indicative of mitochondrial membrane depolarization in a dose-dependent manner. In comparison, control cells emitted mitochondrial red fluorescence with less green fluorescence (Figure 7A). Western blot analysis showed that inhibition of Aurora kinase with VX-680 for 24 hr and 48 hr induced amounts of cleaved caspase-3 expression. The cleavage of the PARP polymerase, a major target for caspases, was also detected in VX-680 treated cells. At dose of 5 nM, cleaved caspase-3 and PARP expression was dramatically increased in NB4-R2 cells (Figure 7B). Interestingly, VX-680-induced activation of caspase pathway was correlated with down-regulation of Akt-1 phosphorylation at the activation site, Ser473 and decreased the level of phosphorylated GSK-3β at Ser9, the downstream of Akt-1 (Figure 7B). Thus, VX-680 suppressed Akt-1 activation, reduced mitochondrial membrane potentials and induced NB4-R2 cells apoptosis by activation of caspase pathway.