A small molecule inhibitor against kinase domain of mammalian target of rapamycin (mTOR) causes autophagic cell death in glioblastoma

3365 Background: Autophagy is a process of intracellular bulk degradation whereby cellular proteins, organelles and cytoplasmic components are engulfed in double membrane forming autophagic vesicles, and delivered to the lysosomal system for subsequent degradation. Autophagy is regulated by phosphotidylinositol-3-kinases (PI3K) and mammalian target of rapamycin (mTOR) pathways. Activation of mTOR kinase has been shown to down-regulate autophagy process. Previous data indicated that autophagy could be induced by rapamycin as well as mTOR small interfering RNA (siRNA). Silencing of mTOR with siRNA augmented the cytotoxicity of rapamycin in glioblastoma, indicating that autophagy was a primary mediator of the anti-tumor effect of rapamycin rather than a protective response. The goals of this study were to determine whether a small molecule inhibitor against the kinase domain of mTOR would cause autophagic cell death. The secondary objective is to develop a high content screen assay for autophagy detection.
 Methods: U-87 MG glioblastoma cells were treated with an mTOR specific kinase inhibitor. Inactivation of both mTOR complexes (TOR1 and TOR2) was evaluated by measuring phosphorylation of S6 Ser 235/236 and AKT Ser473 in cell lysates at indicated times. Autophagy was detected by fluorescent dye monodansylcadaverine (MDC) and acridine orange (AO) and confirmed by high content imaging assay with polyclonal antibody against LC3 (microtubule-associated protein 1 light chain 3), a specific biomarker for autophagy. The cleaved LC3-II protein was measured by western immunoblotting. Cell proliferation was measured by Alamar blue after 72 hours of treatment.
 Results: Upon treatment of U-87 MG cells with mTOR kinase inhibitor for 48 hours, massive autophagy formation was observed in U-87 MG glioblastoma. Using high content imaging assay (Cellomics), the spots in the cytoplasm representing autophagic vesicles were increased in a dose-dependent manner. This correlated with an anti-proliferative effect. We also identified other compounds within the same series that possess a similar effect.
 Conclusion:We demonstrate that an mTOR kinase inhibitor results in significant autophagic cell death in glioblastoma which correlates with the anti-proliferative effect suggesting the blockage of mTOR kinase activity initiates type II programmed cell death.