Caspase-8 activation is necessary but not sufficient for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in the prostatic carcinoma cell line LNCaP.
BACKGROUND
The differential sensitivity of tumor cells to TRAIL-induced apoptosis may be mediated by different intracellular inhibitors of apoptosis, and only a few reports have described the pathway(s) that are activated in response to TRAIL in prostate cells.


METHODS
LNCaP was transfected with a dominant-negative form of FADD (FADD-DN) and cells were selected in the presence of hygromycin. Cell viability was estimated by calcein assay. Apoptosis was estimated by caspase activation using both fluorogenic substrates and Western blot analysis of activated caspases. To detect cytochrome c release, mitochondria-free cytosol was prepared and Western blot analysis was performed.


RESULTS
LNCaP is resistant to TRAIL but TRAIL transiently induces DEVDase activity and activation of caspase-8; caspase-2, -3, -7, and -9 were not activated. Wortmannin, an inhibitor of the PI3K/Akt pathway, converted the phenotype of LNCaP from TRAIL-resistant to -sensitive. In the presence of wortmannin TRAIL induced activation of caspase-2, -3, -7, -8, and -9, as well as dissipation of mitochondrial transmembrane potential and release of cyto-chrome c from mitochondria into the cytosol. In addition, combined TRAIL and wortmannin treatment resulted in cleavage of several proteins: PARP, Akt, p21/WAF1, and MDM2 as well as dephosphorylation of Akt. The proteolysis of p21/WAFI and Akt, which are known survival factors, presumably amplify the apoptotic cascade in LNCaP. Transfection of FADD-DN in LNCaP resulted in inhibition of caspase activation as well as in resistance to combined treatment with TRAIL and wortmannin.


CONCLUSIONS
These results suggest that caspase-8 activation is necessary but not sufficient for TRAIL-mediated apoptosis and is presumably blocked downstream of caspase-8 by the PI3K/Akt pathway.
Rokhlin
Oskar W
OW
Departments of Pathology, The University of Iowa, Iowa City 52242-1087, USA.
Guseva
Natalya V
NV
Tagiyev
Agshin F
AF
Glover
Rebecca A
RA
Cohen
Michael B
MB
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes / Q000494:pharmacology; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000502:physiology; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002934:Cinnamates; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004351:Drug Resistance; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D053200:Fas-Associated Death Domain Protein; D006801:Humans; D006921:Hygromycin B / Q000031:analogs & derivatives* / Q000494:pharmacology; D008297:Male; D008562:Membrane Glycoproteins / Q000494:pharmacology / Q000502:physiology*; D009687:Nuclear Proteins; D010447:Peptide Hydrolases / Q000378:metabolism; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000378:metabolism / Q000473:pathology*; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051736:Proto-Oncogene Proteins c-mdm2; D053221:TNF-Related Apoptosis-Inducing Ligand; D014162:Transfection; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology / Q000502:physiology*; D000077191:Wortmannin
D048868:Adaptor Proteins, Signal Transducing; D000730:Androstadienes; D051017:Apoptosis Regulatory Proteins; C493956:CDKN1A protein, human; D002352:Carrier Proteins; D002934:Cinnamates; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; C505533:FADD protein, human; D053200:Fas-Associated Death Domain Protein; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011506:Proteins; D011518:Proto-Oncogene Proteins; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D006921:Hygromycin B; C026273:hygromycin A; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D010447:Peptide Hydrolases; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; C108711:DEVDase; D000077191:Wortmannin