Selective inhibition of FLICE-like inhibitory protein expression with small interfering RNA oligonucleotides is sufficient to sensitize tumor cells for TRAIL-induced apoptosis.
BACKGROUND
Most tumors express death receptors and their activation represents a potential selective approach in cancer treatment. The most promising candidate for tumor selective death receptor-activation is tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo2L, which activates the death receptors TRAIL-R1 and TRAIL-R2, and induces apoptosis preferentially in tumor cells but not in normal tissues. However, many cancer cells are not or only moderately sensitive towards TRAIL and require cotreatment with irradiation or chemotherapy to yield a therapeutically reasonable apoptotic response. Because chemotherapy can have a broad range of unwanted side effects, more specific means for sensitizing tumor cells for TRAIL are desirable. The expression of the cellular FLICE-like inhibitory protein (cFLIP) is regarded as a major cause of TRAIL resistance. We therefore analyzed the usefulness of targeting FLIP to sensitize tumor cells for TRAIL-induced apoptosis.


METHODS
To selectively interfere with expression of cFLIP short double-stranded RNA oligonucleotides (small interfering RNAs [siRNAs]) were introduced in the human cell lines SV80 and KB by electroporation. Effects of siRNA on FLIP expression were analyzed by Western blotting and RNase protection assay and correlated with TRAIL sensitivity upon stimulation with recombinant soluble TRAIL and TRAIL-R1- and TRAIL-R2-specific agonistic antibodies.


RESULTS
FLIP expression can be inhibited by RNA interference using siRNAs, evident from reduced levels of FLIP-mRNA and FLIP protein. Inhibition of cFLIP expression sensitizes cells for apoptosis induction by TRAIL and other death ligands. In accordance with the presumed function of FLIP as an inhibitor of death receptor-induced caspase-8 activation, down-regulation of FLIP by siRNAs enhanced TRAIL-induced caspase-8 activation.


CONCLUSIONS
Inhibition of FLIP expression was sufficient to sensitize tumor cells for TRAIL-induced apoptosis. The combination of TRAIL and FLIP-targeting siRNA could therefore be a useful strategy to attack cancer cells, which are resistant to TRAIL alone.
Siegmund
Daniela
D
Institute of Cell Biology and Immunology, University of Stuttgart, Germany.
Hadwiger
Philipp
P
Pfizenmaier
Klaus
K
Vornlocher
Hans-Peter
HP
Wajant
Harald
H
D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; D002352:Carrier Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002461:Cell Line, Transformed; D002470:Cell Survival; D019008:Drug Resistance, Neoplasm; D018274:Electroporation; D005347:Fibroblasts / Q000187:drug effects; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D007624:KB Cells / Q000187:drug effects / Q000473:pathology; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D009841:Oligonucleotides / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology*; D012259:Ribonuclease, Pancreatic / Q000378:metabolism; D053221:TNF-Related Apoptosis-Inducing Ligand; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D051017:Apoptosis Regulatory Proteins; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C506386:CFLAR protein, human; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008024:Ligands; D008164:Luminescent Proteins; D008562:Membrane Glycoproteins; D009841:Oligonucleotides; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053221:TNF-Related Apoptosis-Inducing Ligand; C505653:TNFSF10 protein, human; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; D012259:Ribonuclease, Pancreatic; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases