Abstract
2390
Background. Resistance to apoptosis is mediated by pro-survival Bcl-2 family members and is a major mechanism of therapeutic failure in pancreatic cancer. Obatoclax(GX015-070™, Gemin X) is a novel, small-molecule antagonist of the BH3-bindinggroove of the Bcl-2 protein family. Material and Methods. Human pancreatic cancer cell lines (PANC-1, BxPC-3) were incubated with GX015-070 and/or TRAIL and cell viability or DNA fragmentation was measured by the MTS assay (48 h) or Cell Death Detection ELISA kit (6 h), respectively. Caspase activation and poly(ADP-ribose) polymerase (PARP) cleavage were determined by Western blotting. In GX015-070-treated cells, immunoprecipitation of Bak or Bim proteins was performed and Bcl-xL, Bcl-2 or Mcl-1 was probed by Western blotting. Bak and Bax activation were determined by conformation-specific antibodies. Results. GX015-070 (0-5 µM) monotherapy significantly reduced the viability of PANC-1 and BxPC-3 cell lines. GX015-070 induced apoptosis, as shown by a DNA fragmentation assay, and cleaved caspase-8 and -3 only in BxPC-3 cells. Co-administration of GX015-070 (0-2.5 µM) and TRAIL (0-10 ng/ml) synergistically enhanced TRAIL-mediated cytotoxicity and activated caspase-8, -3, Bid and PARP by cleavage in both cell lines. Furthermore, GX015-070 was shown to release Bak from its sequestration by Mcl-1 or Bcl-xL, and to displace Bim from Bcl-2 or Mcl-1 in both cell lines. GX015-075 also enhanced TRAIL-mediated Bax activation as shown by its conformational change, and the combination of GX015-070 and TRAIL induced a Bak conformational change. Conclusion. GX015-070 treatment unsequesters Bim and Bak from Bcl-2 proteins and enhances Bak and Bax activation to significantly augment TRAIL-mediated apoptosis. Together, these findings demonstrate important anti-tumor mechanisms for this novel BH3 mimetic agent, and support its combination with TRAIL for the treatment of pancreatic cancer.
99th AACR Annual Meeting-- Apr 12-16, 2008; San Diego, CA
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