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Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8.

Chacko AD, Liberante F, Paul I, Longley DB, Fennell DA - BMC Cancer (2010)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Queen's University Belfast, Centre for Cancer Research and Cell Biology, Belfast, UK.

ABSTRACT

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.

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VDAC1 silencing blocks cleavage of caspase-8 to its p18 subunit. (A) Confocal microscopy image showing colocalisation of Caspase-8 (green) with the mitochondrial marker protein COX-4 (red) in H460 cells. Colocalisation is indicated by yellow staining in merged image. (B) Caspase-8 western blot of mitochondrial and cytosolic fractions of cells transiently transfected with NT siRNA or VDAC1 siRNA. Catalytically active caspase-8-p18 appears in NT-siRNA control, but not VDAC1-siRNA cells, following TRAIL. Caspase-8-p18 is predominantly in mitochondrial fraction, but excluded from cyotsolic fraction. COX4 blot is shown as mitochondrial marker; α-tubulin is shown as cytosolic marker. (C) Caspase-8 western blot on mitochondrial fraction from H460 shRNA clones treated with 10 ng/ml TRAIL for 24 h. Appearance of 18 kDa form of caspase-8 is strong in mitochondrial fraction of sh-NT control cells but is abolished in sh-VDAC1-1B and sh-VDAC1-2A cell lines. COX4 is shown as mitochondrial loading control. (D) Caspase-8 western blot of cell lysates from MOR cell line transfected with NT siRNA or VDAC1 siRNA. Caspase-8-p18 appears in NT-siRNA control, but not VDAC1-siRNA cells, following 10 ng/ml TRAIL treatment. (E) Caspase-8 western blot of cell lysates from SKMES cell line transfected with NT siRNA or VDAC1 siRNA. Increase in caspase-8 activation to p43 isoform is detected in NT-siRNA cells following 50 ng/ml TRAIL, but not in VDAC1-siRNA cells. Caspase-8 p18 was not detectable in SKMES cell line at reasonable concentrations of TRAIL.
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Figure 2: VDAC1 silencing blocks cleavage of caspase-8 to its p18 subunit. (A) Confocal microscopy image showing colocalisation of Caspase-8 (green) with the mitochondrial marker protein COX-4 (red) in H460 cells. Colocalisation is indicated by yellow staining in merged image. (B) Caspase-8 western blot of mitochondrial and cytosolic fractions of cells transiently transfected with NT siRNA or VDAC1 siRNA. Catalytically active caspase-8-p18 appears in NT-siRNA control, but not VDAC1-siRNA cells, following TRAIL. Caspase-8-p18 is predominantly in mitochondrial fraction, but excluded from cyotsolic fraction. COX4 blot is shown as mitochondrial marker; α-tubulin is shown as cytosolic marker. (C) Caspase-8 western blot on mitochondrial fraction from H460 shRNA clones treated with 10 ng/ml TRAIL for 24 h. Appearance of 18 kDa form of caspase-8 is strong in mitochondrial fraction of sh-NT control cells but is abolished in sh-VDAC1-1B and sh-VDAC1-2A cell lines. COX4 is shown as mitochondrial loading control. (D) Caspase-8 western blot of cell lysates from MOR cell line transfected with NT siRNA or VDAC1 siRNA. Caspase-8-p18 appears in NT-siRNA control, but not VDAC1-siRNA cells, following 10 ng/ml TRAIL treatment. (E) Caspase-8 western blot of cell lysates from SKMES cell line transfected with NT siRNA or VDAC1 siRNA. Increase in caspase-8 activation to p43 isoform is detected in NT-siRNA cells following 50 ng/ml TRAIL, but not in VDAC1-siRNA cells. Caspase-8 p18 was not detectable in SKMES cell line at reasonable concentrations of TRAIL.

Mentions: Caspase-8 localises to mitochondria in various cell lines, where it can undergo further processing and activation [14-18]. Using confocal microscopy, we examined localisation of caspase-8 in H460 cells. Caspase-8 partially colocalised with the mitochondrial marker COX-4 (Figure 2A). Colocalisation was most evident in the perinuclear region, although cytosolic foci of caspase-8 were visible outwith the mitochondrial COX-4 stained area. We therefore examined whether VDAC1 expression affects caspase-8 processing in the mitochondrial compartment. H460 cells were transfected with VDAC1 siRNA or non-targeting (NT) siRNA, incubated with TRAIL for 24 h, and mitochondrial and cytosolic fractions extracted. Activation of caspase-8 involves processing of the 53 kDa pro-caspase zymogen, and intermediate forms, to the catalytically active p18 form. Following TRAIL treatment, activation of caspase-8-p18 protein was observed in the mitochondrial fraction from NT control cells, but this was abolished by VDAC1 silencing (Figure 2B). The active caspase-8-p18 protein was concentrated in the mitochondrial fraction. Cleavage of caspase-8 to p18 was also significantly reduced following TRAIL in the sh-VDAC1 knockdown cells, whereas caspase-8 p18 was strongly detected in the mitochondrial fraction of sh-NT control cells (Figure 2C).


Voltage dependent anion channel-1 regulates death receptor mediated apoptosis by enabling cleavage of caspase-8.

Chacko AD, Liberante F, Paul I, Longley DB, Fennell DA - BMC Cancer (2010)

VDAC1 silencing blocks cleavage of caspase-8 to its p18 subunit. (A) Confocal microscopy image showing colocalisation of Caspase-8 (green) with the mitochondrial marker protein COX-4 (red) in H460 cells. Colocalisation is indicated by yellow staining in merged image. (B) Caspase-8 western blot of mitochondrial and cytosolic fractions of cells transiently transfected with NT siRNA or VDAC1 siRNA. Catalytically active caspase-8-p18 appears in NT-siRNA control, but not VDAC1-siRNA cells, following TRAIL. Caspase-8-p18 is predominantly in mitochondrial fraction, but excluded from cyotsolic fraction. COX4 blot is shown as mitochondrial marker; α-tubulin is shown as cytosolic marker. (C) Caspase-8 western blot on mitochondrial fraction from H460 shRNA clones treated with 10 ng/ml TRAIL for 24 h. Appearance of 18 kDa form of caspase-8 is strong in mitochondrial fraction of sh-NT control cells but is abolished in sh-VDAC1-1B and sh-VDAC1-2A cell lines. COX4 is shown as mitochondrial loading control. (D) Caspase-8 western blot of cell lysates from MOR cell line transfected with NT siRNA or VDAC1 siRNA. Caspase-8-p18 appears in NT-siRNA control, but not VDAC1-siRNA cells, following 10 ng/ml TRAIL treatment. (E) Caspase-8 western blot of cell lysates from SKMES cell line transfected with NT siRNA or VDAC1 siRNA. Increase in caspase-8 activation to p43 isoform is detected in NT-siRNA cells following 50 ng/ml TRAIL, but not in VDAC1-siRNA cells. Caspase-8 p18 was not detectable in SKMES cell line at reasonable concentrations of TRAIL.
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Figure 2: VDAC1 silencing blocks cleavage of caspase-8 to its p18 subunit. (A) Confocal microscopy image showing colocalisation of Caspase-8 (green) with the mitochondrial marker protein COX-4 (red) in H460 cells. Colocalisation is indicated by yellow staining in merged image. (B) Caspase-8 western blot of mitochondrial and cytosolic fractions of cells transiently transfected with NT siRNA or VDAC1 siRNA. Catalytically active caspase-8-p18 appears in NT-siRNA control, but not VDAC1-siRNA cells, following TRAIL. Caspase-8-p18 is predominantly in mitochondrial fraction, but excluded from cyotsolic fraction. COX4 blot is shown as mitochondrial marker; α-tubulin is shown as cytosolic marker. (C) Caspase-8 western blot on mitochondrial fraction from H460 shRNA clones treated with 10 ng/ml TRAIL for 24 h. Appearance of 18 kDa form of caspase-8 is strong in mitochondrial fraction of sh-NT control cells but is abolished in sh-VDAC1-1B and sh-VDAC1-2A cell lines. COX4 is shown as mitochondrial loading control. (D) Caspase-8 western blot of cell lysates from MOR cell line transfected with NT siRNA or VDAC1 siRNA. Caspase-8-p18 appears in NT-siRNA control, but not VDAC1-siRNA cells, following 10 ng/ml TRAIL treatment. (E) Caspase-8 western blot of cell lysates from SKMES cell line transfected with NT siRNA or VDAC1 siRNA. Increase in caspase-8 activation to p43 isoform is detected in NT-siRNA cells following 50 ng/ml TRAIL, but not in VDAC1-siRNA cells. Caspase-8 p18 was not detectable in SKMES cell line at reasonable concentrations of TRAIL.
Mentions: Caspase-8 localises to mitochondria in various cell lines, where it can undergo further processing and activation [14-18]. Using confocal microscopy, we examined localisation of caspase-8 in H460 cells. Caspase-8 partially colocalised with the mitochondrial marker COX-4 (Figure 2A). Colocalisation was most evident in the perinuclear region, although cytosolic foci of caspase-8 were visible outwith the mitochondrial COX-4 stained area. We therefore examined whether VDAC1 expression affects caspase-8 processing in the mitochondrial compartment. H460 cells were transfected with VDAC1 siRNA or non-targeting (NT) siRNA, incubated with TRAIL for 24 h, and mitochondrial and cytosolic fractions extracted. Activation of caspase-8 involves processing of the 53 kDa pro-caspase zymogen, and intermediate forms, to the catalytically active p18 form. Following TRAIL treatment, activation of caspase-8-p18 protein was observed in the mitochondrial fraction from NT control cells, but this was abolished by VDAC1 silencing (Figure 2B). The active caspase-8-p18 protein was concentrated in the mitochondrial fraction. Cleavage of caspase-8 to p18 was also significantly reduced following TRAIL in the sh-VDAC1 knockdown cells, whereas caspase-8 p18 was strongly detected in the mitochondrial fraction of sh-NT control cells (Figure 2C).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Queen's University Belfast, Centre for Cancer Research and Cell Biology, Belfast, UK.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus