Figure 1.
Effects of tozasertib and alisertib on neuroblastoma cell viability.
A) IC50 values determined after 120 h of incubation by MTT assay; B) tozasertib IC50 values in the presence of the ABCB1 inhibitor zosuquidar (5 µM); C) Average IC50 values in high and low ABCB1-expressing cells, * P<0.05 compared to low ABCB1-expressing cells; D) IC50 values in UKF-NB-3 cells, UKF-NB-3 cells transduced with a lentiviral vector encoding for ABCB1 (UKF-NB-3ABCB1), and UKF-NB-3 cells transduced with a control vector (UKF-NB-3control), * P<0.05 compared to UKF-NB-3.
Figure 2.
Tozasertib (1 µM)-induced expression of p53 target genes as indicated by qPCR.
Expression levels are presented as fold change relative to non-treated controls. * P<0.05 relative to non-treated control.
Figure 3.
Role of p53 signalling in response to aurora kinase inhibition.
A) tozasertib-induced p53 and p21 expression as indicated by Western blot after 24 h of incubation; B) tozasertib- and alisertib-induced expression of p53 target genes in UKF-NB-3 cells in which p53 was depleted using a lentiviral vector encoding shRNA directed against p53 (UKF-NB-3p53shRNA) or in UKF-NB-3 cells transduced with a control vector encoding non-targeting scramble shRNA (UKF-NB-3scr) as indicated by qPCR after 24 h. Expression levels are presented as fold change relative to non-treated controls. * P<0.05 relative to non-treated control; C) tozasertib and alisertib concentrations that reduce UKF-NB-3, UKF-NB-3scr, and UKF-NB-3p53shRNA viability by 50% (IC50). * P<0.05 relative to UKF-NB-3 cells.
Figure 4.
Effects of tozasertib on the viability of neuroblastoma cells in combination with the MDM2 inhibitor nutlin-3.
Neuroblastoma cells were treated for five days with tozasertib, nutlin-3, or their combination. Cell viability was determined by MTT assay. The drug concentrations were: UKF-NB-3, tozasertib 6 nM, Nutlin-3 0.625 µM; IMR-32, tozasertib 6 nM, Nutlin-3 1.25 µM; UKF-NB-3rCDDP1000, tozasertib 6 nM, Nutlin-3 1.25 µM; UKF-NB-3rDOX20, tozasertib 156 nM, Nutlin-3 2.5 µM; UKF-NB-3rNutlin10µM, tozasertib 156 nM, Nutlin-3 5 µM; UKF-NB-3rVCR10, tozasertib 156 nM, Nutlin-3 5 µM. * P<0.05 relative to either single treatment.
Figure 5.
Effects of tozasertib on histone H3 phosphorylation (indicating aurora kinase A and B activity), cell cycle distribution, and apoptosis (indicated by BAX activation) in neuroblastoma cells.
A) Numbers of cells expressing phosphorylated histone H3 were determined flow cytometry; * P<0.05 relative to untreated control; B) Representative histograms indicating cell cycle distribution in neuroblastoma cells after tozasertib treatment, arrows indicate additional peaks with high DNA content, possibly indicating endoreduplication; C) Numbers of cells with activated BAX expressed as fold change relative to control as determined by flow cytometry using an antibody specific for activated BAX after 24 h of tozasertib treatment. * P<0.05 relative to untreated control.
Figure 6.
Tozasertib-induced apoptosis in UKF-NB-3 cells transduced with a lentiviral vector encoding shRNA directed against p53 (UKF-NB-3p53shRNA) and UKF-NB-3 transduced with a control vector expressing non-targeting scrambled shRNA (UKF-NB-3scr) as indicated by BAX activation, caspase 3/7 activation, and determination of sub-G1 cells.
* P<0.05 relative to UKF-NB-3scr.