PI3K/mTORC1/2 inhibitor PQR309 inhibits proliferation and induces apoptosis in human glioblastoma cells

Abstract

Glioblastoma (GBM) is the most common type of primary central nervous system tumor in adults, which has high mortality and morbidity rates, and short survival time, namely <15 months after the diagnosis and application of standard therapy, which includes surgery, radiation therapy and chemotherapy; thus, novel therapeutic strategies are imperative. The activation of the PI3K/AKT signaling pathway plays an important role in GBM. In the present study, U87 and U251 GBM cells were treated with the PI3K/mTORC1/2 inhibitor PQR309, and its effect on glioma cells was investigated. Cell Counting Kit‑8 assay, 5‑ethynyl‑2'‑deoxyuridine and colony formation assays revealed dose‑ and time‑dependent cytotoxicity in glioma cells that were treated with PQR309. Flow cytometry and western blotting revealed that PQR309 can significantly induce tumor cell apoptosis and arrest the cell cycle in the G1 phase. Furthermore, the expression levels of AKT, phosphorylated (p)‑AKT, Bcl‑2, Bcl‑xL, Bad, Bax, cyclin D1, cleaved caspase‑3, MMP‑9 and MMP‑2 were altered. In addition, the migration and invasion of glioma cells, as detected by wound healing, migration and Transwell invasion assays, exhibited a marked suppression after treating the cells with PQR309. These results indicated that PQR309 exerts an antitumor effect by inhibiting proliferation, inducing apoptosis, inducing G1 cell cycle arrest, and inhibiting invasion and migration in human glioma cells. The present study provides evidence supportive of further development of PQR309 for adjuvant therapy of GBM.

Keywords: PQR309; glioblastoma; apoptosis; proliferation; invasion.

Figure 1.

(A) Molecular structure of PQR309. (B) Cell viability for U87 and U251 cells after being treated with PQR309 with various concentrations. (C) The colony formation rates of U87 and U251 cells in various concentration groups. Scale bar, 50 µm. (D) Each cell line was treated at different time-points with the IC₅₀ values of PQR309 for the CCK-8 assay. Each cell line was analyzed in triplicate.

Figure 2.

The nucleus was stained with Hoechst 33342 (blue) and the proliferative cells were stained red with EdU. Scale bar, 20 µm. The two cell lines were treated with various concentrations of PQR309 for 72 h. (A and C) The percentage of Edu-positive cells after being treated with PQR309. (B and D) The total percentage of stained nuclei in cells treated with PQR309 (*P<0.05).

Figure 3.

Cell cycle arrest assay. (A and B) Cell cycle distribution analysis by flow cytometry of U87 and U251 cells after being treated with various concentrations of PQR309 (0–20 µM) for 72 h. (C and D) The percentage of the cells in G1 and S+G2 phases of U87 and U251 cells.

Figure 4.

AKT, p-AKT (ser 473), cyclin D1, MMP-2 and MMP-9 expression in U87 and U251 cells after treatment with PQR309. A representative experiment is presented. Each protein was analyzed in triplicate.

Figure 5.

Wound closure assay. (A and B) Cell lines were treated with PQR309 for 48 h and the untreated controls with 0 µM of PQR309. Scale bar, 50 µm. (C and D) The rate of wound closure of U87 and U251 cells after being treated with PQR309. *P<0.05.

Figure 6.

Migration and invasion abilities of U87 and U251 cells after treatment with PQR309. (A and C) The migration and the invasion abilities of glioblastoma cells. after being treated with PQR309 at various concentrations. (B and D) Quantification of the results of A and C. *P<0.05 and **P<0.01.

Figure 7.

PQR309 induces apoptosis in U87 and U251 cells stained with 7-ADD/PE. (A and B) Apoptosis of U87 and U251 cells treated with increasing concentrations of PQR309 for 72 h. (C and D) Apoptosis rates of U87 and U251 cells treated with different concentrations of PQR309. *P<0.05 and **P<0.01.

Figure 8.

ΔΨm of (A) U87 cells (treated with 5 µ) and (B) U251 cells (treated with 10 µ) after being treated with PQR309 according to the manufacturer's protocol.

Figure 9.

Expression of Bcl-2, Bcl-xL, Bax, Bad and cleaved caspase-3 and GAPDH in U87 and U251 cells after being treated with PQR309 with various concentrations (0, 5, 10, 20 µM).

Figure 10.

TUNNEL trail of U87 and U251 cells (B) after being treated with PQR309 with IC₅₀ concentrations and (A) without any treatment, respectively.