Effects of cIAP-1, cIAP-2 and XIAP triple knockdown on prostate cancer cell susceptibility to apoptosis, cell survival and proliferation.
BACKGROUND
Manipulating apoptotic resistance represents an important strategy for the treatment of hormone refractory prostate cancer. We hypothesised that the Inhibitor of Apoptosis (IAP) Proteins may be mediating this resistance and knockdown of cIAP-1, cIAP-2 and XIAP would increase sensitivity to apoptosis.


METHODS
cIAP-1, cIAP-2 and XIAP where knocked down either individually or in combination using siRNA in androgen independent prostate cancer PC-3 cells as confirmed by real-time PCR and western blotting. Cells were then treated with TRAIL, Etoposide, or Tunicamycin, and apoptosis assessed by PI DNA staining. Apoptosis was confirmed with Annexin V labelling and measurement of PARP cleavage, and was inhibited using the pan-caspase inhibitor, zVAD.fmk. Clonogenic assays and assessment of ID-1 expression by western blotting were used to measure recovery and proliferation.


RESULTS
PC-3 are resistant to TRAIL induced apoptosis and have elevated expression of cIAP-1, cIAP-2 and XIAP. Combined knockdown sensitised PC-3 to TRAIL induced apoptosis, but not to Etoposide or Tunicmycin, with corresponding increases in caspase activity and PARP cleavage which was inhibited by ZVAD.fmk. Triple knock down decreased proliferation which was confirmed by decreased ID-1 expression.


CONCLUSIONS
Simultaneous knock down of the IAPs not only sensitised the PC-3 to TRAIL but also inhibited their proliferation rates and clonogenic survival. The inability to alter sensitivity to other triggers of apoptosis suggests that this effect is specific for death receptor pathways and knock down might facilitate immune-surveillance mechanisms to counter cancer progression and, in combination with therapeutic approaches using TRAIL, could represent an important treatment strategy.
Gill
Catherine
C
UCD School of Medicine and Medical Science, University College Dublin, Dublin, Ireland. cgill@hrb.ie
Dowling
Catherine
C
O'Neill
Amanda J
AJ
Watson
R William G
RW
D017209:Apoptosis / Q000187:drug effects; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D003627:Data Interpretation, Statistical; D005047:Etoposide / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics* / Q000378:metabolism; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism* / Q000473:pathology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000494:pharmacology; D014415:Tunicamycin / Q000494:pharmacology; D044767:Ubiquitin-Protein Ligases; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics / Q000378:metabolism
D051034:Inhibitor of Apoptosis Proteins; D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; D051636:X-Linked Inhibitor of Apoptosis Protein; C496866:XIAP protein, human; D014415:Tunicamycin; D005047:Etoposide; C494832:BIRC3 protein, human; D000075747:Baculoviral IAP Repeat-Containing 3 Protein; D044767:Ubiquitin-Protein Ligases; D010447:Peptide Hydrolases; C108711:DEVDase