Aurora B kinase inhibitor AZD1152: determinants of action and ability to enhance chemotherapeutics effectiveness in pancreatic and colon cancer

Abstract

Background: AZD1152, the prodrug for AZD1152-hydroxyquinazoline pyrazol anilide (HQPA), is a selective inhibitor of Aurora B kinase activity. Preclinical evaluation of AZD1152 has been reported in several human cancer models. The potentiality of this compound in combination therapy warrants further investigation in solid tumours.

Experimental design: This study explored the effects of AZD1152-HQPA in colon and pancreatic tumour cells. The antitumour properties of AZD1152, either as single agent or in combination with chemotherapeutics, were evaluated in each study model. The efficacy and the toxicity of AZD1152 alone and in combination with gemcitabine were validated in pancreatic tumour xenograft model.

Results: AZD1152-HQPA treatment resulted in a dramatic increase of chromosome number, modification of cell cycle and induction of apoptosis. The most effective combination was that with chemotherapeutics given soon after AZD1152 in both tumour cell types. The effectiveness of the sequential schedule of AZD1152 with gemcitabine was confirmed in nude mice bearing MiaPaCa-2 tumours, showing inhibition of tumour volumes and delaying of tumour growth after the interruption of the treatments. Here we show that AZD1152-HQPA enhances oxaliplatin and gemcitabine effectiveness in colon and pancreatic cancer, respectively. First, we provide advances into administration schedules and dosing regimens for the combination treatment in in vivo pancreatic tumour.

Figure 1

Drug(s)-dependent cell growth inhibition. Cells were incubated with AZD1152-HQPA (AZD) and/or oxaliplatin (oxa) or gemcitabine (gem) with continuous or intermittent exposure. Cell survival was determined by cell counts. In (A) and (B), results from MiaPaCa-2, and MiaPaCa-2 and HCT116. 1D WO=1 day drug washout; 3D WO=3 day drug washout.

Figure 2

AZD1152-HQPA-dependent cell cycle modification. Cells were incubated with AZD1152-HQPA (AZD) for 1 day, followed by 0–3 days drug wash out, and then the cell cycle was analysed by CFM as described in Material and Methods section. In (A) and (B), results from AZD-treated HCT116 and MiaPaCa-2 cells, followed by 0, 1 and 3 days drug wash out were reported. In (C), results from 3-day AZD-treated MiaPaCa-2 cells are shown in the function of concentration. FL2-A=FL2-area (total cell fluorescence).

Figure 3

Morphological changes by AZD1152-HQPA exposure. (A) Increase in MiaPaCa-2 cell size induced by AZD1152-HQPA exposure at various concentrations and wash out. (B) Chromosome alignment and segregation prevented by AZD1152-HQPA in MiaPaCa-2 cells induced abnormal prometaphase detected by immunofluorescence (tubulin staining in green and DAPI in blue). (C) MiaPaCa-2 cells were incubated with AZD1152-HQPA for 1 day and the chromosome number was determined by quinacrine assay utilising fluorescence microscopy, as described in Materials and Methods section. A number of at least 50 metaphases for each specimen were evaluated.

Figure 4

Effect of AZD1152-HQPA in combination with oxaliplatin on cell growth and apoptosis. HCT116 cells were seeded in 96-well plates. After 24 h, cells were subjected to concurrent dosing schedule (A and B) or a sequential dosing schedule (C). Cells were then stained for cell number by Hoechst and BAK-epitope, an early marker of apoptosis. Images were taken and end points quantified using the Cellomics Arrayscan II platform. Graphs are representative of three independent experiments. Columns, mean; bars, s.e. ^*P<0.05 vs oxaliplatin 0 μM (two-way ANOVA); WO=washout.

Figure 5

AZD1152-HQPA-dependent modulation of cell pathways. (A) MiaPaCa-2 cells were incubated with AZD1152-HQPA for 1 day, followed by 0 or 3 days drug wash out, and then histone 3 phosphorylation was analysed by immunofluorescence staining (Ser-10 pH3 staining in red and DAPI in blue) and western blotting. (B and C) Cells were incubated with AZD1152-HQPA and protein extracts were analysed by western blotting. The amount of p-AKT and p53 was determined using β-actin to normalise values.

Figure 6

In vivo effects of AZD1152, gemcitabine and the sequential combination of AZD1152, followed by gemcitabine on MiaPaCa-2 tumour xenografts. (A) Antitumor effect of AZD1152, gemcitabine and their consecutive combination on MIAPaCa-2 tumours xenotransplanted in CD nu/nu mice. The mice were randomised into groups of eight animals. (B) Body weight of MIAPaCa-2 tumour-bearing control mice and mice treated with metronomic AZD1152, gemcitabine and their consecutive combination. Symbols and bars, mean±s.e.m.; ^*P<0.05 vs vehicle-treated controls.