Background: The Phosphatidylinositol 3- Kinase (PI3 Kinase) pathway is implicated in resistance of tumours to radiotherapy. In this study we tested the hypothesis that NVP-BEZ235, a potent novel dual inhibitor of PI3 Kinase and mammalian Target of Rapamycin (mTOR), would enhance the therapeutic effects of ionizing radiation in vitro. Methods: The effects of NVP-BEZ235 were evaluated in cell lines with activation of the PI3 Kinase pathway: H460 (a non small cell lung cancer (NSCLC) cell line with a PIK3CA mutation); A549 (a NSCLC cell line with a KRAS mutation); and the A431 cell line (a squamous cell lung cancer line with amplified EGFR). In vitro radiosensitization was assessed with clonogenic survival assays using BEZ235 at doses of 50 nM and 100 nM and a radiation dose range from 0-8 Gy. Mechanisms of radiosensitization were studied using FACS analysis for DNA content for cell cycle and apoptosis; and gamma H2AX immunostaining and Alkaline COMET assays for quantification of radiation-induced DNA damage and assessment of DNA repair after radiation. Results: Potent cellular radiosensitization was observed with clonogenic survival assays in A549, H460 and A431 cell lines. The D37 (The dose of radiation that results in 37% surviving clonogenic fraction) was reduced from 5.2 Gy to 1.8 Gy and 0.9 Gy in A549 cells; from 4Gy to 2.5 and 1Gy in H460 cells; and from 3.3 Gy to 1.9 Gy and 0.8 Gy after treatment with 50 and 100nm of NVP-BEZ235 respectively. Investigation of mechanisms of radiosensitization demonstrated effects on cell cycle distribution with an increase in cells in G1 following drug treatment and an increase in both G1 and G2/M following drug and radiation treatment. Apoptosis after radiation was enhanced in the H460 cell line and also A549 cells but only with NVP-BEZ235 doses of 1µM . Significantly NVP-BEZ235 reduced repair of DNA damage after radiation.\#947;-H2AX expression persisted at 6 and 24 hours after radiation in cells treated with NVP-BEZ235. The Alkaline COMET assays demonstrated a significant persistence of radiation-induced double strand breaks as measured by Olive tail moment at 6 hours (57.3±2.2 vs 31.0±1.4, p<0.00001) and 24 hours (45.3±1.6 vs 13.8±1.0, p<0.00001) in H460 and at 6 hours(41.6±1.7 vs 33.8±1.8, p=0.001) and 24 hours (42.7±3.6 vs 26.6±1.8, p=0.00009) in A549 cells treated with 250nM of NVP-BEZ235 and 4 Gy radiation (results shown are mean±SEM for cells treated with NVP-BEZ235 vs vehicle controls, p-values are two-sided). Conclusion: NVP-BEZ235 potently enhances the effect of ionizing radiation in a range of epithelial cell lines at physiologically relevant concentrations. Multiple mechanisms including effects on cell cycle distribution, apoptosis, and repair of radiation-induced DNA damage are responsible for in vitro radiosensitization.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2302.

100th AACR Annual Meeting-- Apr 18-22, 2009; Denver, CO

American Association for Cancer Research
2009