BH3 mimetic obatoclax enhances TRAIL-mediated apoptosis in human pancreatic cancer cells.
OBJECTIVE
Prosurvival Bcl-2 proteins inhibit the mitochondrial and death receptor-mediated apoptotic pathways. Obatoclax is a small-molecule antagonist of the BH3-binding groove of Bcl-2 proteins that may enhance tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) sensitivity and efficacy.


METHODS
Human pancreatic cancer cell lines were incubated with obatoclax and/or TRAIL and cell viability, Annexin V labeling, caspase cleavage, and cytochrome c release were measured. In drug-treated cell lines, protein-protein interactions were studied by immunoprecipitation. Bax/Bak activation was analyzed using conformation-specific antibodies. Lentiviral short hairpin RNA was used to knockdown Bim, Bid, and apoptosis-inducing factor (AIF) expression.


RESULTS
Obatoclax reduced the viability of PANC-1 and BxPC-3 cell lines and synergistically enhanced TRAIL-mediated cytotoxicity. Obatoclax enhanced TRAIL-mediated apoptosis, as shown by Annexin V labeling, which was accompanied by caspase activation (caspase-8, -9, and -3) and cleavage of Bid. Obatoclax potentiated TRAIL-mediated Bax/Bak activation and the release of mitochondrial cytochrome c, Smac, and AIF. Mechanisms underlying the apoptotic effect of obatoclax include displacement of Bak from its sequestration by Bcl-x(L) or Mcl-1 and release of Bim from Bcl-2 or Mcl-1. Bid knockdown by short hairpin RNA attenuated caspase cleavage and cytotoxicity of obatoclax plus TRAIL. Bim knockdown failed to inhibit the cytotoxic effect of obatoclax alone or combined with TRAIL yet attenuated TRAIL-mediated cytotoxicity. AIF knockdown attenuated cytotoxicity of the drug combination.


CONCLUSIONS
Obatoclax potentiates TRAIL-mediated apoptosis by unsequestering Bak and Bim from Bcl-2/Bcl-x(L) or Mcl-1 proteins. This drug combination enhances Bid-mediated cross-talk between the mitochondrial and death receptor-mediated apoptotic pathways and may represent a novel therapeutic strategy against pancreatic cancer.
Huang
Shengbing
S
Miles and Shirley Fiterman Center for Digestive Diseases and Division of Oncology, Mayo Clinic, Rochester, Minnesota 55901, USA.
Okumura
Kenji
K
Sinicrope
Frank A
FA
D017209:Apoptosis / Q000187:drug effects; D051023:BH3 Interacting Domain Death Agonist Protein / Q000494:pharmacology; D020169:Caspases / Q000096:biosynthesis; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism; D006801:Humans; D007211:Indoles; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010190:Pancreatic Neoplasms / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011758:Pyrroles / Q000494:pharmacology*; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000378:metabolism; D051028:bcl-2-Associated X Protein / Q000378:metabolism
C494772:BAK1 protein, human; C494826:BAX protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D007211:Indoles; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D011758:Pyrroles; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D050998:bcl-2 Homologous Antagonist-Killer Protein; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D020169:Caspases; C520962:obatoclax