Downregulation of c-FLIP sensitizes DU145 prostate cancer cells to Fas-mediated apoptosis.
Although DU145 prostate cancer cells are resistant to exogenously applied Fas agonist CH-11 (anti-Fas monoclonal antibody), Fas-resistance can be overcome using a FasL expressing adenovirus (AdGFPFasL(TET)) [Hyer et al., Molecular Therapy, 2000; 2:348-58 (ref.12)]. The purpose of this study was to try to understand why DU145 cells are resistant to CH-11 and determine the signaling pathway utilized by AdGFPFasL(TET) to induce apoptosis in these Fas-resistant cells. Using immunoblot analysis, we show that AdGFPFasL(TET) is capable of initiating the classic Fas-mediated apoptotic pathway in DU145 cells, which includes activation of caspases-8, -3, -7, and -9, BID cleavage, cytochrome c release from mitochondria, and PARP cleavage. In contrast, CH-11 binds to Fas, but is unable to transmit the death signal beyond the plasma membrane suggesting a block at the DISC (death inducing signaling complex). The anti-apoptotic protein c-FLIP (cellular Flice-like inhibitory protein), which has been shown to inhibit Fas-mediated apoptosis at the DISC, was down-regulated following AdGFPFasL(TET) treatment prompting us to investigate its role in inhibiting CH-11-induced cell death. Using c-FLIP anti-sense oligonucleotides to down-regulate c-FLIP we sensitized DU145 cells to CH-11-induced apoptosis. These data suggest that c-FLIP may play a critical role in regulating Fas-mediated apoptosis in prostate cancer cells and that modulation of c-FLIP may enhance Fas signaling based therapies.
Hyer
Marc L
ML
Department of Microbiology and Immunology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Sudarshan
Sunil
S
Kim
Youngsoo
Y
Reed
John C
JC
Dong
Jian-yun
JY
Schwartz
David A
DA
Norris
James S
JS
D017209:Apoptosis; D051023:BH3 Interacting Domain Death Agonist Protein; D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins / Q000378:metabolism*; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D016923:Cell Death; D002460:Cell Line; D002469:Cell Separation; D003574:Cytochrome c Group / Q000378:metabolism; D015536:Down-Regulation; D004789:Enzyme Activation; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D019169:Jurkat Cells; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D019014:fas Receptor / Q000378:metabolism*
D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D002352:Carrier Proteins; D003574:Cytochrome c Group; C505661:FASLG protein, human; D053222:Fas Ligand Protein; D047908:Intracellular Signaling Peptides and Proteins; D008562:Membrane Glycoproteins; D016376:Oligonucleotides, Antisense; D019014:fas Receptor; C505407:CASP3 protein, human; C505520:CASP7 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053179:Caspase 7; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases