Combined Bcl 2 /mTOR Inhibition Leads to Enhanced Radiosensitization via Induction of Autophagy and Apoptosis in Non-small Lung Tumor Xenograft Model

Abstract

Radiotherapy has a central role in the treatment of locally advanced non-small cell lung cancer. The effectiveness of this modality, however, is often limited, in part due to the presence of defects in cell death pathways. In this study, we tested whether co-upregulation of autophagy and apoptosis can be utilized to enhance radiation sensitivity of lung cancer cells in vitro and in a lung cancer xenograft model. Clonogenic assay was used to determine radiosensitivity. H460 lung cancer xenografts was generated and immunohistochemistry (IHC) was performed on tumor sections for tumor cell proliferation (Ki67 staining), angiogenesis (von Willebrand factor staining), and apoptosis (cleaved caspase-3 antibody staining). Autophagy was determined by punctate localization of GFP-LC3 fusion protein, which observed under a confocal fluorescence microscope in vitro. Apoptosis measured using the Annexin V-fluorescent isothiocyante apoptosis detection kit I (Pharmingen) with flow cytometry in vitro. Autophagy was induced by rapamycin, an mTOR inhibitor, and apoptosis was induced by AT-737, a Bcl2 inhibitor. The combination of AT-737 and rapamycin markedly enhanced sensitivity of H460 cells to radiation (DER = 2.28, p = 0.001) in clonogenic assay. This enhanced radiosensitization was associated with an increase in characteristic punctate localization of GFP-LC3 and in Annexin-V positive cells, suggesting that both apoptosis and autophagy were induced simultaneously. In addition, the concurrent administration of AT-737, rapamycin and radiation in vivo significantly decreased cell proliferation by 77% (p < 0.001) and vascular density by 67.5% (p < 0.008) compared to radiation alone. Consistent with the in vitro results, the tri-modality treatment showed a significant 14-fold increase in caspase-3 activity as a marker for apoptosis in the lung tumor xenograft model. These results suggest that concurrent upregulation of autophagy and apoptosis may be utilized as a novel strategy to enhance radiation therapy in lung cancer.