Antitumor activity of NVP-BKM120--a selective pan class I PI3 kinase inhibitor showed differential forms of cell death based on p53 status of glioma cells

Abstract

Purpose: The aim of this study was to show preclinical efficacy and clinical development potential of NVP-BKM120, a selective pan class I phosphatidylinositol-3 kinase (PI3K) inhibitor in human glioblastoma (GBM) cells in vitro and in vivo.

Experimental design: The effect of NVP-BKM120 on cellular growth was assessed by CellTiter-Blue assay. Flow cytometric analyses were carried out to measure the cell-cycle, apoptosis, and mitotic index. Mitotic catastrophe was detected by immunofluorescence. The efficacy of NVP-BKM120 was tested using intracranial U87 glioma model.

Results: We tested the biologic effects of a selective PI3K inhibitor NVP-BKM120 in a set of glioma cell lines. NVP-BKM120 treatment for 72 hours resulted in a dose-dependent growth inhibition and effectively blocked the PI3K/Akt signaling cascade. Although we found no obvious relationship between the cell line's sensitivity to NVP-BKM120 and the phosphatase and tensin homolog (PTEN) and epidermal growth factor receptor (EGFR) statuses, we did observe a differential sensitivity pattern with respect to p53 status, with glioma cells containing wild-type p53 more sensitive than cells with mutated or deleted p53. NVP-BKM120 showed differential forms of cell death on the basis of p53 status of the cells with p53 wild-type cells undergoing apoptotic cell death and p53 mutant/deleted cells having a mitotic catastrophe cell death. NVP-BKM120 mediates mitotic catastrophe mainly through Aurora B kinase. Knockdown of p53 in p53 wild-type U87 glioma cells displayed microtubule misalignment, multiple centrosomes, and mitotic catastrophe cell death. Parallel to the assessment of the compound in in vitro settings, in vivo efficacy studies using an intracranial U87 tumor model showed an increased median survival from 26 days (control cohort) to 38 and 48 days (treated cohorts).

Conclusion: Our present findings establish that NVP-BKM120 inhibits the PI3K signaling pathways, leading to different forms of cell death on the basis of p53 statuses. Further studies are warranted to determine if NVP-BKM120 has potential as a glioma treatment.

© 2011 AACR.

Figures

Figure 1

NVP-BKM120 inhibits glioma cell proliferation. A, structure of NVP-BKM120. B, waterfall plot of IC50 micromolar values. These graphs show that NVP-BKM120 has a particular growth inhibition signature, with some cell lines being exquisitely sensitive and others being relatively resistant. For this, glioma cell lines were plated in 96-well plates at a density of 5,000 cells per well. Cells were treated with increasing concentrations of NVP-BKM120 in triplicate wells for 72 hours, and cell viability was assessed by CellTiter-Blue assay as described in Materials and Methods. The results shown are of a single experiment with 3 independent replicates. C, the relative sensitivity of GBM cell lines to the PI3K inhibitor NVP-BKM120 versus p53 and PTEN status. The sensitivity of NVP-BKM120 against the GBM cell lines measured using CellTiter-Blue assay. Indicated values represent the concentration causing 50% growth inhibition (IC50). Values are expressed relative to the mean of IC50 of LN751 cell line using the equation: −log2 (IC50 individual cell line/IC50 of LN751). D, dot blot showing correlation between p53 and PTEN status and in vitro drug sensitivity as shown by the IC50 of individual cell line. Lines indicate median value. The wild-type p53 cells are more sensitive to NVP-BKM120 than p53-mutant/deleted glioma cells (P < 0.0001).

Figure 2

Molecular responses of human glioma cells to NVP-BKM120. A and B, cell lysates were separated by gel electrophoresis and immunoblotted for phospho-Akt (Ser473), phospho-Akt (Thr308), phospho-S6 (Ser240/244 and Ser235/236), and phospho-4E-BP1 (Thr37/46). Total Akt, total S6, total 4E-BP1, and actin were used as loading controls. C, representative cell-cycle profiles of glioma cells following 72 hours exposure to IC50 concentration of NVP-BKM120 represented as stacked columns (*, P < 0.04; **, P < .0001; #, P < 0.0001; ##, P < 0.0001). D, accumulation of multinucleated cells in U251 cells exposed to increasing concentrations of NVP-BKM120. Scale bars, 25 μm. E, quantification of multinucleated cells after NVP-BKM120 in glioma cells. Con, control.

Figure 3

Mitotic catastrophe cell death on NVP-BKM120 treatment. Seventy-two hours after treatment with 2 μmol/L of NVP-BKM120, cells were collected and immunoassayed with anti-α-F-actin antibody counterstained with 4′,6-diaminidino-2-phenylindole. A, mitotic catastrophe figures showing multiple centrosomes, predominant in U251 cell line. Scale bars, 50 μm. B, significant increase in mitotic entry on treatment with NVP-BKM120 in p53-mutated cell lines. Mean ± SD of 3 independent experiments. Mitotic index in control untreated cells and cells after 72 hours of incubation with PI3K inhibitor is represented. C, Western blot showing p-Aurora B decrease in U251 cells after NVP-BKM120 treatment in a time-dependent manner. β-Actin is used as a loading control. Con, control.

Figure 4

NVP-BKM120 treatment induces apoptosis in glioma cells containing mutant p53. A, glioma cell lines were treated with 1 and 2 μmol/L NVP-BKM120 for 72 hours before they were harvested for Annexin V staining as an indication of apoptosis. The population of cells positive for Annexin V staining was compiled as shown. U251, LN18, and LN229 cell lines had minimum apoptosis, whereas U87, D54, and U343 cell lines produced maximum Annexin V-positive staining. B, glioma cells were treated with 2 μmol/L NVP-BKM120 for various time intervals, as indicated. Total protein was extracted and subjected for the detection of PARP cleavage and cleaved caspase-3 in a Western blot analysis using an anti-PARP and anti-caspase-3 antibody. Results showed that PARP and caspase-3 were cleaved after NVP-BKM120 treatment in U87 but not in U251 cells.

Figure 5

p53 knockdown restores mitotic catastrophe in U87 glioma cells. A, cell lysates were separated by gel electrophoresis and immunoblotted for p53, p-Aurora B, and Aurora B, and actin was used as loading control. B, quantification of multinucleated cells after NVP-BKM120 in glioma cells. C, p53 knockdown U87 cells were treated with 2 μm of NVP-BKM120; cells were collected and immunoassayed with anti-α-F-actin antibody counterstained with 4′,6-diaminidino-2-phenylindole. Scale bars, 50 μm. D, significant increase in mitotic entry as determined by p-H3-positive cells on treatment with NVP-BKM120 in p53 knockdown U87 cell line. Mean ± SD of 3 independent experiments. Mitotic index in control untreated cells and cells after 72 hours of incubation with PI3K inhibitor is represented.

Figure 6

NVP-BKM120 extends survival in an intracranial animal model. A, U87 cells were implanted intracranially in nude mice (n = 10 in each group), and treatment was commenced 4 days later. NVP-BKM120 (20 and 40 mg/kg/d) was administered orally 5 times a week for a total of 20 treatments. The control group mice were treated with NMP/PEG300. Mice were sacrificed at morbidity, and survival curves were compared by using Prism 5 software. The treatment of NVP-BKM120 showed a statistically significant improvement over control (P = 0.001 for 20 mg/kg P = 0.0006 for 40 mg/kg), as determined by pair-wise log-rank Mantel–Cox test of the Kaplan–Meier survival curves. B, immunoblotting analyses of both the expression and activation of Akt and S6 in 2-week and 4-week tumors following NVP-BKM120 treatment. Immunoblotting analyses showed that NVP-BKM120 inhibited the activity of Akt and S6 in both sets of tumors, as assessed by the level of corresponding phosphorylated protein. C, analysis of NVP-BKM120 effects on tumor xenograft proliferation. Nude athymic mice with established U87 xenografts were treated with vehicle control and NVP-BKM120 (40 mg/kg), tumors were harvested, formalin fixed, and stained for Ki-67 as a marker of proliferation. Tumor sections were stained with antibodies PI3K downstream target p-Akt, apoptotic markers cleaved PARP, and cleaved caspase-3 as described in Materials and Methods. Representative microscopic fields of each immunohistochemical reaction with the overall semiquantitative grading scale. Scale bars, 25 μm. Con, control.