PI3Kβ inhibitor AZD6482 exerts antiproliferative activity and induces apoptosis in human glioblastoma cells

Abstract

Glioblastoma is the most common type of primary brain tumour in adults, and its pathogenesis is particularly complicated. Among the many possible mechanisms underlying its pathogenesis, hyperactivation of the PI3K/Akt pathway is essential to the occurrence and development of glioma through the loss of PTEN or somatic activating mutations in PIK3CA. In the present study, we investigated the effect of the PI3Kβ inhibitor AZD6482 on glioma cells. The CCK-8 assay showed dose-dependent cytotoxicity in glioma cell lines treated with AZD6482. Additionally, AZD6482 treatment was found to significantly induce apoptosis and cell cycle arrest as detected using flow cytometry. Moreover, as shown using western blot analysis, the levels of p-AKT, p-GSK-3β, Bcl-2, and cyclin D1 were decreased after AZD6482 treatment. In addition, we found that AZD6482 inhibited the migration and invasion of glioma cells as detected by wound healing and Transwell invasion assays. Taken together, our findings indicate that AZD6482 exerts an antitumour effect by inhibiting proliferation and inducing apoptosis in human glioma cells. AZD6482 may be applied as an adjuvant therapy to improve the therapeutic efficacy of glioblastoma treatment.

Figure 1.

PTEN is frequently mutated in glioblastoma, and cancer cell lines with PTEN mutations were sensitive to AZD6482. (A) Mutation data of glioblastoma from The Cancer Genome Atlas (TCGA) database showed the genetic alteration status of the PI3K signalling pathway. (B) Data of GDSC database showed the relationship between various gene mutations and drug sensitivity. Scatter plot showed IC₅₀ values of PIK3CA-mutated, PIK3CB-mutated, PTEN-mutated and corresponding wild-type cell lines following treatment with AZD6482. Each point represents the IC₅₀ value for an individual cell line. The red lines indicate the geometric mean. The lower and upper lines indicated the minimum and maximum concentration of AZD6482. Mut, mutated; wild, wild-type. *P<0.05, **P<0.01

Figure 2.

Treatment with AZD6482 leads to an anti-proliferative effect on glioblastoma cells. (A) The molecular structure of AZD6482. (B) Cell viability was determined by CCK-8 assay after AZD6482 treatment at various concentrations (0, 0.625, 1.25, 2.5, 5, 10, 20, or 40 µM) for 48 h. Each cell line was analysed in triplicate. (C) AZD6482 inhibited the colony formation of U87 and U118 cells. Fewer colonies were formed in the treated group compared with the control group. Scale bar, 50 µm. (D) DNA replication activity was assessed by an EdU incorporation assay. Nuclei were stained with Hoechst (blue), and the proliferative cells were dyed red with EdU. Scale bar, 100 µm (same magnification in all panels).

Figure 3.

AZD6482 induces apoptosis in U87 and U118 cells. (A) U87 and U118 cells were treated with increasing concentrations of AZD6482 for 48 h, which was followed by an analysis of apoptosis by staining with 7-ADD/PE. (B) Apoptotic cells were measured by flow cytometry. **P<0.01, compared with that of untreated controls (0 µM). Data are presented as the mean ± SD of at least three independent experiments. The expression levels of (C) Bcl-2, Bax and (D) related proteins of the PI3K signalling pathway were used to assess the inhibitory effect of AZD6482. Each protein was analysed in triplicate, and a representative experiment is shown.

Figure 4.

AZD6482 induces cell cycle arrest at the G1 phase in U87 cells. (A) Cell cycle distribution analysis by flow cytometry. AZD6482-induced G1 phase arrest in U87 cells after exposure to AZD6482 (0–40 µM) for 48 h. (B) The percentage of the cell population in G1/G0, S and G2/M phases. Data are presented as the mean ± SD of at least three independent experiments (n=3). **P<0.01, ***P<0.001, compared with the untreated controls (0 µM). (C) The effects of AZD6482 on the expression of cyclin D1 are presented by western blot analysis.

Figure 5.

AZD6482 inhibits glioblastoma cell migration and invasion. (A) The invasion ability of U87 and U118 gradually decreased with increasing concentrations of AZD6482. Scale bar, 100 µm. (B) The invaded cells were counted under a light microscope. (C) The cell monolayer was wounded with a pipette tip and treated with 0 or 10 µM AZD6482. Images were acquired at 0, 24, and 48 h in the wound-healing assay. Scale bar, 500 µm. (D) The rate of wound closure in the AZD6482-treated group was lower than that in the untreated group at 48 h of growth. Data are presented as the means ± SD of three independent experiments; *P<0.05, **P<0.01, ***P<0.001, compared with the untreated controls (0 µM).