The BH3 mimetic ABT-737 potentiates TRAIL-mediated apoptosis by releasing Bim and Bak proteins in human pancreatic cancer cells

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Background. TRAIL has been shown to induce mitochondrial apoptotic signaling that can be negatively regulated by pro-survival Bcl-2 proteins. ABT-737 is a small molecule BH3 mimetic that binds to and antagonizes Bcl-2, Bcl-x_L, but not Mcl-1.
 Materials and Methods. Human pancreatic cancer cell lines (PANC-1, BxPC-3) were incubated with ABT-737 (Abbott) and/or TRAIL and cell viability or DNA fragmentation was measured by the MTS assay (48 h) or Cell Death Detection ELISA kit (4 h), respectively. Caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage were determined by Western blotting. In ABT-737-treated cells, immunoprecipitation of Bak or Bim proteins was performed, and Bcl-x_L, Bcl-2 or Mcl-1 were probed by Western blotting. Bax activation was determined by a conformation-specific antibody.
 Results. ABT-737 (10, 20 µM) monotherapy was shown to reduce cell viability. Co-administration of ABT-737 (0-20 µM) and TRAIL (0-10 ng/ml) was found to significantly potentiate a TRAIL-mediated reduction in cell viability and an induction of apoptosis (shown by a DNA fragmentation assay), in BxPC-3 and PANC-1 cell lines. ABT-737 enhanced TRAIL-induced activation of caspase-8, Bid, and downstream caspase-3 and PARP cleavage. ABT-737 enhanced a Bax conformational change induced by TRAIL. Furthermore, ABT-737 released Bak from its sequestration by Bcl-x_L in both cell lines. ABT-737 also displaced the pro-apoptotic BH3-only protein Bim from its sequestration by Bcl-x_L or Bcl-2. Bim shRNA was shown to attenuate caspase-3 cleavage and to reduce the cytotoxic effects of ABT-737 plus TRAIL, compared to shRNA control cells. Finally, Mcl-1 shRNA potentiated caspase-3 cleavage by ABT-737 and enhanced its cytotoxic effects.
 Conclusion. ABT-737 can potentiate TRAIL-mediated apoptosis by displacing Bim and Bak from Bcl-x_L and/or Bcl-2, and can enhance Bax activation by TRAIL. These findings suggest a novel strategy to enhance cross-talk between the extrinsic and intrinsic apoptotic pathways to improve therapeutic efficacy against pancreatic cancer.