Abstract #374: BH3 mimetic, ABT-737, enhances celecoxib-induced apoptosis that can be further augmented by inhibition of autophagy in human colon cancer cells.

Background: Celecoxib is a selective inhibitor of the cyclooxygenase-2 (COX-2) enzyme and has been shown to prevent or regress colorectal neoplasms in humans. Celecoxib induces apoptosis in colon cancer cells and we explored strategies to enhance cell death including use of the BH3 mimetic agent, ABT-737, as well as an inhibitor of autophagy. Autophagy is protein degradation process whereby cellular proteins and organelles are delivered to lysosomes for digestion. Many anti-neoplastic drugs induce autophagy and suppression of autophagy may enhance apoptosis induction. Materials & Methods: SW480 human colon cancer cells were stably transfected with a Bcl-2 plasmid. Human colon cancer cell lines (SW480 or HT-29, HCT116 wild-type and Bax knockout) were incubated with celecoxib (0-120 µM) alone and in combination with ABT-737 (0-5 µM; Abbott), in the presence or absence of 3-methyladenine (3-MA; 10 mM). Caspase cleavage, Bid truncation, and LC3I, II expression were determined using highly specific antibodies by Western blotting. A GFP-LC3B chimeric plasmid was introduced into HT-29 cells and fluorescence microscopy was performed. Bcl-x_L or Beclin 1 knockdown HT-29 cells were generated using lentiviral short hairpin RNA (shRNA). Results: Cells with ectopic Bcl-2 expression or Bax knockout displayed resistance to celecoxib-induced apoptosis, while knockdown of Bcl-x_L proteins sensitized cells to apoptosis-induced by celecoxib. Furthermore, ABT-737 synergistically enhanced the cytotoxicity of celecoxib, and apoptosis induction by this drug combination was associated with cleavage of caspases -8,-9,-3 and truncation of Bid. ABT-737 combined with celecoxib induced the conversion of autophagosome-associated protein light chain 3 (LC3) from cytosolic (LC3I) to membrane-bound (LC3II) forms. ABT-737 plus celecoxib also increased autophagic cells, detected by GFP-LC3 using FACS analysis, compared to either drug alone. Addition of 3-methyladenine (3-MA), an inhibitor of autophagosome formation and a class III PI3 kinase inhibitor, blocked LC3 induction by celecoxib alone or combined with ABT-737. Furthermore, 3-MA enhanced apoptosis induction by celecoxib and/or ABT-737, as shown by cleavage of caspases-8,-9, -3 and truncated Bid expression. Moreover, the addition of 3-MA produced a 5-fold increase in apoptosis induced by celecoxib plus ABT-737, as shown by Annexin V labeling. Conclusion: ABT-737 synergistically enhances celecoxib-induced cell death. Furthermore, apoptosis induction by the combination of celecoxib and ABT-737 can be markedly enhanced by pharmacologic inhibition of autophagy, suggesting a novel therapeutic strategy to enhance apoptosis induction in colon cancers.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 374.