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Bid participates in genotoxic drug-induced apoptosis of HeLa cells and is essential for death receptor ligands' apoptotic and synergistic effects.

Köhler B, Anguissola S, Concannon CG, Rehm M, Kögel D, Prehn JH - PLoS ONE (2008)

Bottom Line: While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin.Similar effects were observed using the Bid inhibitor BI6C9.They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.

ABSTRACT

Background: The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases.

Methodology/principal findings: To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations.

Conclusions/significance: Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.

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Bid depletion abrogates the death receptor-mediated apoptotic pathway.Control and HeLa Bid kd were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) for 1 h followed by treatment for the specified times with CHX (1 µg/ml) in combination with an agonistic Fas antibody or recombinant TRAIL at the indicated concentrations. Controls were treated with vehicle. A, B) Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl). C, D) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E, F) Cells were treated as indicated and apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).
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pone-0002844-g002: Bid depletion abrogates the death receptor-mediated apoptotic pathway.Control and HeLa Bid kd were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) for 1 h followed by treatment for the specified times with CHX (1 µg/ml) in combination with an agonistic Fas antibody or recombinant TRAIL at the indicated concentrations. Controls were treated with vehicle. A, B) Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl). C, D) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E, F) Cells were treated as indicated and apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).

Mentions: We next compared the effect of the bid knockdown to death receptor-induced apoptosis triggered by the activating Fas antibody or by treatment with recombinant human TRAIL (TR). Dose-response analyses demonstrated that the knockdown of bid potently reduced DEVD cleavage activity in response to both stimuli compared to a control clone transfected with a scrambled sequence (Fig. 2 A, B). Procaspase-3 processing, poly-ADP-ribose polimerase (PARP) cleavage and phosphatidylserine exposure were analyzed by Western blotting and flow cytometry (Fig. 2 C, D, E, F). The kinetics of procaspase-3 processing corresponded with the generation of the caspase-cleaved 85 kDa PARP-fragment, the latter being detectable 2 h after TRAIL/cycloheximide (Chx) and 4 h after Fas/Chx incubation. In HeLa Bid kd cells, procaspase-3 processing, cleaved PARP levels, and the accumulation of Annexin V-positive cells were significantly reduced. Importantly, the knockdown of bid also significantly increased the clonogenic survival of HeLa cells in response to TRAIL and Fas activation as analyzed in colony formation assays (Fig. 3 A, B, C, D).


Bid participates in genotoxic drug-induced apoptosis of HeLa cells and is essential for death receptor ligands' apoptotic and synergistic effects.

Köhler B, Anguissola S, Concannon CG, Rehm M, Kögel D, Prehn JH - PLoS ONE (2008)

Bid depletion abrogates the death receptor-mediated apoptotic pathway.Control and HeLa Bid kd were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) for 1 h followed by treatment for the specified times with CHX (1 µg/ml) in combination with an agonistic Fas antibody or recombinant TRAIL at the indicated concentrations. Controls were treated with vehicle. A, B) Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl). C, D) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E, F) Cells were treated as indicated and apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2481399&req=5

pone-0002844-g002: Bid depletion abrogates the death receptor-mediated apoptotic pathway.Control and HeLa Bid kd were pre-incubated with the pan-caspase inhibitor zVAD-fmk (100 µM) for 1 h followed by treatment for the specified times with CHX (1 µg/ml) in combination with an agonistic Fas antibody or recombinant TRAIL at the indicated concentrations. Controls were treated with vehicle. A, B) Caspase-3 like activity was measured by cleavage of the fluorogenic substrate Ac-DEVD-AMC. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl). C, D) Cells were treated as indicated and lysates were subjected to Western blotting with a polyclonal caspase-3, a monoclonal Poly-ADP-Ribose Polymerase (PARP) antibody and a monoclonal α-tubulin antibody. E, F) Cells were treated as indicated and apoptosis was assessed by flow cytometric evaluation of Annexin-V FITC conjugated binding to phosphatidylserine in non-permeabilized cells. Data are means+/−SD from n = 3 separate experiments. # p<0.05 difference from control cells (Ctrl).
Mentions: We next compared the effect of the bid knockdown to death receptor-induced apoptosis triggered by the activating Fas antibody or by treatment with recombinant human TRAIL (TR). Dose-response analyses demonstrated that the knockdown of bid potently reduced DEVD cleavage activity in response to both stimuli compared to a control clone transfected with a scrambled sequence (Fig. 2 A, B). Procaspase-3 processing, poly-ADP-ribose polimerase (PARP) cleavage and phosphatidylserine exposure were analyzed by Western blotting and flow cytometry (Fig. 2 C, D, E, F). The kinetics of procaspase-3 processing corresponded with the generation of the caspase-cleaved 85 kDa PARP-fragment, the latter being detectable 2 h after TRAIL/cycloheximide (Chx) and 4 h after Fas/Chx incubation. In HeLa Bid kd cells, procaspase-3 processing, cleaved PARP levels, and the accumulation of Annexin V-positive cells were significantly reduced. Importantly, the knockdown of bid also significantly increased the clonogenic survival of HeLa cells in response to TRAIL and Fas activation as analyzed in colony formation assays (Fig. 3 A, B, C, D).

Bottom Line: While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin.Similar effects were observed using the Bid inhibitor BI6C9.They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland.

ABSTRACT

Background: The BH3-only protein Bid is an important component of death receptor-mediated caspase activation. Bid is cleaved by caspase-8 or -10 into t-Bid, which translocates to mitochondria and triggers the release of caspase-activating factors. Bid has also been reported to be cleaved by other proteases.

Methodology/principal findings: To test the hypothesis that Bid is a central mediator of stress-induced apoptosis, we investigated the effects of a small molecule Bid inhibitor on stress-induced apoptosis, and generated HeLa cells deficient for Bid. Stable knockdown of bid lead to a pronounced resistance to Fas/CD95- and TRAIL-induced caspase activation and apoptosis, and significantly increased clonogenic survival. While Bid-deficient cells were equally sensitive to ER stress-induced apoptosis, they showed moderate, but significantly reduced levels of apoptosis, as well as increased clonogenic survival in response to the genotoxic drugs Etoposide, Oxaliplatin, and Doxorubicin. Similar effects were observed using the Bid inhibitor BI6C9. Interestingly, Bid-deficient cells were dramatically protected from apoptosis when subtoxic concentrations of ER stressors, Etoposide or Oxaliplatin were combined with subtoxic TRAIL concentrations.

Conclusions/significance: Our data demonstrate that Bid is central for death receptor-induced cell death and participates in anti-cancer drug-induced apoptosis in human cervical cancer HeLa cells. They also show that the synergistic effects of TRAIL in combination with either ER stressors or genotoxic anti-cancer drugs are nearly exclusively mediated via an increased activation of Bid-induced apoptosis signalling.

Show MeSH
Related in: MedlinePlus