X-linked inhibitor of apoptosis (XIAP) blocks Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis of prostate cancer cells in the presence of mitochondrial activation: sensitization by overexpression of second mitochondria-derived activator of caspase/direct IAP-binding protein with low pl (Smac/DIABLO).
The resistance to Apo2 ligand (Apo2L)/tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis could be overcome by treatment with subtoxic concentrations of actinomycin D (Act D) in prostate tumor cells. Furthermore, the sensitization to Apo2L/TRAIL-mediated apoptosis by Act D positively correlated with selective down-regulation of X-linked inhibitor of apoptosis (XIAP). In this study, we examined whether second mitochondria-derived activator of caspase/direct inhibitor of apoptosis-binding protein with low pl (Smac/DIABLO), a known inhibitor of apoptosis (IAP)-neutralizing protein, sensitizes resistant prostate tumor cells to Apo2L/TRAIL-mediated apoptosis. The prostate tumor cell line CL-1 was treated with Apo2L/TRAIL, Act D, or a combination of the two. The apoptosis-mediated signaling pathway was examined by Western blotting and flow cytometry. Furthermore, CL-1 cells transfected with the anti-IAP inhibitor Smac/DIABLO were examined for sensitivity to Apo2L/TRAIL. Whereas Apo2L/TRAIL induced the release of cytochrome c and endogenous Smac/DIABLO in the CL-1 tumor cells, the cytosolic levels of both molecules were not sufficient to induce apoptosis. Transient transfectants with a Smac/DIABLO cDNA encoding a neutralizing inhibitor of IAPs were sensitized to Apo2L/TRAIL-mediated apoptosis. The sensitization to Apo2L/TRAIL by Smac/DIABLO overexpression was a result of synergistic activation of caspases-3, -9, and -8. Treatment of the Smac/DIABLO transient transfectant with Apo2L/TRAIL enhanced the release of Smac/DIABLO from mitochondria and led to reduction of IAP family proteins (XIAP, c-IAP1, and c-IAP2). These results show that Smac/DIABLO can sensitize CL-1 tumor cells to Apo2L/TRAIL-mediated apoptosis. Thus, up-regulation of Smac/DIABLO and sensitization to Apo2L/TRAIL-mediated apoptosis are of potential clinical application in the immunotherapy of drug-/Apo2L/TRAIL-resistant tumors.
Ng
Chuen-Pei
CP
Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles School of Medicine, Jonsson Comprehensive Cancer Center, University of California, Los Angeles, California 90095, USA.
Bonavida
Benjamin
B
D000911:Antibodies, Monoclonal / Q000378:metabolism; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002455:Cell Division; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D053938:DNA Fragmentation; D018076:DNA, Complementary / Q000378:metabolism; D003609:Dactinomycin / Q000494:pharmacology; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007167:Immunotherapy; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008297:Male; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D024101:Mitochondrial Proteins / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000378:metabolism; D011506:Proteins / Q000494:pharmacology / Q000502:physiology*; D015398:Signal Transduction; D053221:TNF-Related Apoptosis-Inducing Ligand; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000737:chemistry* / Q000378:metabolism*; D015854:Up-Regulation; D051636:X-Linked Inhibitor of Apoptosis Protein
D000911:Antibodies, Monoclonal; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D003574:Cytochrome c Group; C412403:DIABLO protein, human; D018076:DNA, Complementary; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008562:Membrane Glycoproteins; D024101:Mitochondrial Proteins; D011506:Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D003609:Dactinomycin; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases