Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8.
BACKGROUND
Activation of the extrinsic apoptosis pathway by tumour necrosis factor related apoptosis inducing ligand (TRAIL) is a novel therapeutic strategy for treating cancer that is currently under clinical evaluation. Identification of molecular biomarkers of resistance is likely to play an important role in predicting clinical anti tumour activity. The involvement of the mitochondrial type 1 voltage dependent anion channel (VDAC1) in regulating apoptosis has been highly debated. To date, a functional role in regulating the extrinsic apoptosis pathway has not been formally excluded.


METHODS
We carried out stable and transient RNAi knockdowns of VDAC1 in non-small cell lung cancer cells, and stimulated the extrinsic apoptotic pathway principally by incubating cells with the death ligand TRAIL. We used in-vitro apoptotic and cell viability assays, as well as western blot for markers of apoptosis, to demonstrate that TRAIL-induced toxicity is VDAC1 dependant. Confocal microscopy and mitochondrial fractionation were used to determine the importance of mitochondria for caspase-8 activation.


RESULTS
Here we show that either stable or transient knockdown of VDAC1 is sufficient to antagonize TRAIL mediated apoptosis in non-small cell lung cancer (NSCLC) cells. Specifically, VDAC1 is required for processing of procaspase-8 to its fully active p18 form at the mitochondria. Loss of VDAC1 does not alter mitochondrial sensitivity to exogenous caspase-8-cleaved BID induced mitochondrial depolarization, even though VDAC1 expression is essential for TRAIL dependent activation of the intrinsic apoptosis pathway. Furthermore, expression of exogenous VDAC1 restores the apoptotic response to TRAIL in cells in which endogenous VDAC1 has been selectively silenced.


CONCLUSIONS
Expression of VDAC1 is required for full processing and activation of caspase-8 and supports a role for mitochondria in regulating apoptosis signaling via the death receptor pathway.
Chacko
Alex D
AD
Queen's University Belfast, Centre for Cancer Research and Cell Biology, Belfast, UK.
Liberante
Fabio
F
Paul
Ian
I
Longley
Daniel B
DB
Fennell
Dean A
DA
D017209:Apoptosis; D015153:Blotting, Western; D053446:CASP8 and FADD-Like Apoptosis Regulating Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D002289:Carcinoma, Non-Small-Cell Lung / Q000378:metabolism* / Q000473:pathology*; D053181:Caspase 8 / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D008175:Lung Neoplasms / Q000378:metabolism* / Q000473:pathology*; D008928:Mitochondria / Q000378:metabolism / Q000473:pathology; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering / Q000494:pharmacology; D053218:Receptors, Death Domain / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D053221:TNF-Related Apoptosis-Inducing Ligand / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D014407:Tumor Cells, Cultured; D050995:Voltage-Dependent Anion Channel 1 / Q000378:metabolism*; D019014:fas Receptor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
D053446:CASP8 and FADD-Like Apoptosis Regulating Protein; C491901:FAS protein, human; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D053218:Receptors, Death Domain; D053221:TNF-Related Apoptosis-Inducing Ligand; C494762:VDAC1 protein, human; D019014:fas Receptor; D050995:Voltage-Dependent Anion Channel 1; D053181:Caspase 8