Abstract 4858: Celecoxib-induces a Bcl-2/Bcl-xL-regulated autophagy whose inhibition drives human colon cancer cells into apoptosis

Abstract

Abstract BACKGROUND. Autophagy is a mechanism of cellular destruction whereby cytoplasmic proteins and organelles are sequestered in vacuoles and delivered to lysosomes for degradation and recycling. Autophagy is not always pro-death but can also be pro-survival under conditions of cellular stress. Autophagy and apoptosis can be regulated by Bcl-2 family proteins. We determined whether the anti-neoplastic agent celecoxib can induce autophagy and/or apoptosis, and if a small molecule Bcl-2/Bcl-xL antagonist, e.g., ABT-737, can enhance either/both processes. Furthermore, we determined whether autophagy inhibition can drive colon cancer cells into apoptosis. EXPERIMENTAL METHODS. Human colon cancer cell lines (HCT116, SW480 ± Bcl-2, HT-29 ± Bcl-xL lentiviral shRNA) were incubated with celecoxib (0-120 µM) alone and in combination with ABT-737 (0- 5 µM). Autophagy was studied by LC3I/II expression (Western blotting) and using a GFP-LC3B chimeric plasmid. Additionally, autophagy was imaged by monodansylcadaverine (MDC) staining of autophagic vacuoles and acridine orange staining of acidic vesicles by fluorescent confocal microscopy. Apoptosis was analyzed by caspase cleavage (Western blotting) by annexin V labeling and FACS. RESULTS. Celecoxib triggered conversion of the autophagosome-associated protein light chain 3 (LC3) from a cytosolic (LC3I) to a membrane-bound (LC3II) form as shown by Western blotting, and a change in the localization of the fluorescence pattern of an ectopic GFP-LC3B protein. Celecoxib-induced conversion of LC3 was due to autophagy induction, as shown using the lysosome inhibitor, Bafilomycin A1 that produced an accumulation of LC3II. Celecoxib-induced autophagy and apoptosis were attenuated by ectopic Bcl-2, but were augmented by Bcl-xL knockdown. ABT-737 was shown to enhance celecoxib-induced autophagy as shown by LC3 conversion, acridine orange staining of acidic vesicles, and MDC staining of autophagic vacuoles. Celecoxib also induced apoptosis, as shown by caspase cleavage and annexin V labeling, that was synergistically enhanced by ABT-737. Given that autophagy can be pro-survival under conditions of cellular stress, we determined whether inhibition of autophagy can drive cells into apoptosis. Use of the autophagy inhibitor 3-methyladenine (3-MA) was shown to block LC3 conversion and to enhance celecoxib-induced apoptosis that was further augmented by ABT-737. Similarly, Vps34 or Atg8/LC3 siRNA were shown to enhance caspase cleavage in cells treated with celecoxib plus ABT-737. CONCLUSION. Celecoxib induces autophagy and apoptosis that can both be potentiated by a Bcl-2/ Bcl-xL antagonist. Furthermore, inhibition of autophagy was shown to drive cells into apoptosis, suggesting a novel therapeutic strategy against colon cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4858.