BKM120 sensitizes C6 glioma cells to temozolomide via suppression of the PI3K/Akt/NF-κB/MGMT signaling pathway

Oncol Lett. 2017 Dec;14(6):6597-6603. doi: 10.3892/ol.2017.7034. Epub 2017 Sep 22.

Abstract

Glioblastoma is the most common type of malignant intracranial tumor in adults. Temozolomide (TMZ), as the first-line chemotherapy agent used in patients with glioblastoma, has demonstrated different effects in patients due to the expression of O6-methylguanine-DNA methyltransferase (MGMT) which is able to repair the DNA lesions induced by TMZ. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is over-activated in glioblastoma and has been revealed to be potentially implicated in resistance to TMZ. BKM120, a selective pan class I PI3K inhibitor, has been reported to facilitate apoptosis and reverse drug resistance in advanced solid tumors. The present study aims to investigate whether BKM120 is able to sensitize glioma cells to TMZ. C6 glioma cells were treated with BKM120 and/or TMZ for 12, 24 and 48 h, respectively. Cell Counting Kit-8 assays were performed to determine cell viability. The level of apoptosis was evaluated by Hoechst 33342 and TUNEL staining, and the levels of cleaved caspase-3 and Bcl-2-like protein 4 (Bax) expression was measured. Furthermore, the present study investigated the possible mechanism underlying BKM120 reverse chemoresistance to TMZ. The downstream targets of PI3K, including phosphorylated (p)-Akt, nuclear factor (NF)-B p65, were analyzed by western blotting. The MGMT transcription levels in monotherapy and combination therapy were demonstrated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The present study revealed that monotherapy treatments with either drug required a high concentration found reduction in cell viability. However, a low concentration of BKM120 inhibited the PI3K/Akt/NF-κB signaling pathway activity in glioma cells and significantly increased TMZ cytotoxicity. The coefficient of drug interaction was ~0.7. Results from the TUNEL assay, Hoechst 33342 staining and evaluation of the levels of cleaved caspase-3 and Bax expression also confirmed the finding that the combination treatment induced a higher level of apoptosis compared with the TMZ monotherapy. RT-qPCR demonstrated that the combination strategy reversed the TMZ-induced MGMT over-transcription. The reduction of NF-κB p65 in combination treatment supported the hypothesis that BKM120 may mediate MGMT transcription via inhibition of NF-κB p65. In conclusion, BKM120 and TMZ demonstrated strong synergistic cytotoxicity in C6 glioma cells. The BKM120-induced NF-κB p65 inhibition may be involved in the mediation of MGMT transcription to reverse TMZ-resistance in C6 glioma cells.

Keywords: BKM120; O6-methylguanine-DNA methyltransferase; apoptosis; chemotherapy; glioma; nuclear factor-κB; temozolomide.