Combining Radiotherapy with PI3K Isoform-Selectivity Inhibitor Increases Radiosensitivity And Suppresses Tumor Growth In Glioblastoma Cells

Abstract

Glioblastoma (GBM) is the most malignant cancer that occurs within the brain and shows the dismal prognosis. The phosphoinositide 3-kinase (PI3K)-AKT signaling pathway plays a critical role in GBM. Also, the activation of Akt due to irradiation induces radioresistance. But, the pan-PI3K inhibitors have side-effects in clinical trial. Different PI3K isoforms play non-redundant roles in particular tumor. So, it is expected that selective inhibition of PI3K isoforms decrease side effects. Therefore, we investigated whether combining radiotherapy with the PI3K isoform inhibitor decreases radioresistance and tumor growth in GBM. Glioma 261 expressed luciferase (GL261-luc) cell lines were used to identify the effects of combination therapy. GL261-luc were irradiated 1Gy with or without PI3K isoform inhibitors. After irradiation, clonogenic assay was performed to confirm the GBM survival rate and observed inhibition of PI3K-AKT signaling pathway using western blot (WB) assay. In order to confirm to repair DNA double strand breaks (DSB), the expression of phospho-H2A.X was confirmed by immunofluorescence. Also, to observe the effects of combination therapy in GBM in vivo, an orthotopic mouse model was established by intracranial injection implanting GL261-luc. Irradiation was performed specifically to brain with 2Gy, 3Gy and 4Gy for 5 days with or without PI3K-α isoform inhibitor. We observed tumor growth using IVIS image and analyzed splenocyte RNA and protein level using RT-PCR and WB. According to inhibition of PI3K-AKT signaling, GL261-luc radiosensitivity was significantly increased. GL261-luc irradiated by 1Gy was suppressed cell growth about 70% compared to control (p<0.001). When irradiated with PI3K-isform inhibitor, each survival rate was about 18% (PI3K-pan, p<0.00001), 25% (PI3K-α, p<0.00001), 30% (PI3K-δ, p<0.00001), 45% (PI3K-γ/δ, p<0.00001). Also, the expression of phospho-H2A.X, DNA DSB marker, was increased by combination therapy. Also, GL261-luc irradiated with PI3K- isoform inhibitor was decreased the ability of migration. Especially, the effects of combining radiotherapy with PI3K-α inhibitor was similar as much as with PI3K-pan inhibitor (In vitro). So, we selected PI3K-α inhibitor and analyzed combination therapy effects in GBM in vivo. We demonstrated that combining radiotherapy with the PI3K-α isoform inhibitor was markedly delayed tumor growth than without inhibitor (p<0.0001). In addition, the expression of PD1, regulator of cancer immune system, in splenocyte was increased by combination therapy (p<0.001) (In vivo). Our findings demonstrate that combining radiotherapy with the PI3K-α isoform increased radiosensitivity. Also, GBM tumor growth was markedly suppressed and increased immunogenic marker such as PD1. Combining radiotherapy with the PI3K-α isoform inhibitor would be a potential strategy for overcoming radioresistance in GBM, but further study should be necessary.