2806 PI3K/mTOR pathway inhibitors enhance radiosensitivity in radiation resistant oral squamous cell carcinomas by inducing cell cycle arrest

Abstract

Background: Radiation therapy is often used to treat oral cancer but toxicity and local control remain problematic. PI3K and mTOR activation has been linked to decreased radiation responsiveness in oral cancer, so it limits efficacy of radiotherapy. In this study, we invertigated the NVPBEZ235, a dual PI3K/mTOR inhibitor in rediosensitization of OSCC cell lines and a radiation-resistant cell line. Additionally, we investigated further the effect of RAD001, NVP-BEZ235 on the primary OSCC tumors. Methods: We developed a radioresistant oral cancer cells (OML1-R), from the certain oral cancer patient in Taiwan by repeated exposure to step-wise fractionated ionizing radiation. We performed clonogenic survival assays on OSCC cell lines (SCC4, SCC25), OML1-R and exposed to increasing dose of radiation (0 to 4 Gy) in the presence of RAD001 and NVP-BEZ235. Analysis of target protein phosphorylation and cell cycle were performed. We also isolated four different primary cancer cells from tumor tissues of individual OSCC patients and examined the effects of radiation alone and in combination with NVP-BEZ235 by the clonogenic survival assay. Results: Combined treatment of the NVP-BEZ235 with radiation effectively inhibited phosphorylation of AKT, S6, 4EBP1 and eIF4E target proteins and reduced clonogenic survival in OSCC cell lines (SCC4 and SCC25) and OML1-R. Similarly, NVP-BEZ235 also sensitized all of the four primary OSCC cells to radiation. The treatment arrested OSCC cell lines, OML1-R and primary OSCC cells at G1 and G2/M phases of the cell cycle. NVPBEZ235 treatment reduced the protein levels of cyclin D1and CDK4 and also changed the protein levels of CDK inhibitors, p27 and p21 leading to G1 cell cycle delay. Besides, NVP-BEZ235 enhanced the cytotoxic effect of radiation on OSCC cells without inducing significant cell apoptotic and did not affect H2AX phosphorylation, as a marker of DNA damage. Conclusions: The present study identified radiosensitizing effect of NVPBEZ235 in oral cancer cells and primary tumors and the effect is due to arrest in both G1 and G2 phases of cell cycle when treatments are combined, suggesting that BEZ235 may be as a potential therapeutic strategy for improving clinical outcome in OSCC. No conflict of interest.