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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 is necessary for the synergistic activity between genotoxic drugs and TRAIL.A, B) HeLa Control and HeLa Bid kd cells were treated with Etoposide (10 µM) or Oxaliplatin (30 µg/ml) for the indicated times; the pan-caspase inhibitor zVAD (100 µM) was added to the cells 1 h prior to treatment where specified; cell lysates were subjected to Western blotting with a polyclonal DR4, a polyclonal DR5, and a monoclonal β-actin antibody. C, D) HeLa control and HeLa Bid kd cells were pre-incubated with Etoposide (10 µM), Oxaliplatin (30 µg/ml), or vehicle for 16 h followed by treatment with TRAIL (10 ng/ml) for 3 h. 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 Etoposide, Oxaliplatin or TRAIL individual treatments. # p<0.05 difference from control cells (Ctrl).
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pone-0002844-g010: Bid is necessary for the synergistic activity between genotoxic drugs and TRAIL.A, B) HeLa Control and HeLa Bid kd cells were treated with Etoposide (10 µM) or Oxaliplatin (30 µg/ml) for the indicated times; the pan-caspase inhibitor zVAD (100 µM) was added to the cells 1 h prior to treatment where specified; cell lysates were subjected to Western blotting with a polyclonal DR4, a polyclonal DR5, and a monoclonal β-actin antibody. C, D) HeLa control and HeLa Bid kd cells were pre-incubated with Etoposide (10 µM), Oxaliplatin (30 µg/ml), or vehicle for 16 h followed by treatment with TRAIL (10 ng/ml) for 3 h. 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 Etoposide, Oxaliplatin or TRAIL individual treatments. # p<0.05 difference from control cells (Ctrl).

Mentions: We next investigated the role of Bid in the potential synergism between genotoxic drugs and TRAIL. Oxaliplatin and Etoposide both upregulated DR5 receptor expression in a time-dependent manner, while DR4 levels remained constant (Fig. 10 A, B). Similar to our data obtained with ER stressors, we observed potent synergistic effects of Oxaliplatin and Etoposide with TRAIL which was completely abolished in Bid-deficient cells (Fig. 10 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 is necessary for the synergistic activity between genotoxic drugs and TRAIL.A, B) HeLa Control and HeLa Bid kd cells were treated with Etoposide (10 µM) or Oxaliplatin (30 µg/ml) for the indicated times; the pan-caspase inhibitor zVAD (100 µM) was added to the cells 1 h prior to treatment where specified; cell lysates were subjected to Western blotting with a polyclonal DR4, a polyclonal DR5, and a monoclonal β-actin antibody. C, D) HeLa control and HeLa Bid kd cells were pre-incubated with Etoposide (10 µM), Oxaliplatin (30 µg/ml), or vehicle for 16 h followed by treatment with TRAIL (10 ng/ml) for 3 h. 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 Etoposide, Oxaliplatin or TRAIL individual treatments. # 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-g010: Bid is necessary for the synergistic activity between genotoxic drugs and TRAIL.A, B) HeLa Control and HeLa Bid kd cells were treated with Etoposide (10 µM) or Oxaliplatin (30 µg/ml) for the indicated times; the pan-caspase inhibitor zVAD (100 µM) was added to the cells 1 h prior to treatment where specified; cell lysates were subjected to Western blotting with a polyclonal DR4, a polyclonal DR5, and a monoclonal β-actin antibody. C, D) HeLa control and HeLa Bid kd cells were pre-incubated with Etoposide (10 µM), Oxaliplatin (30 µg/ml), or vehicle for 16 h followed by treatment with TRAIL (10 ng/ml) for 3 h. 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 Etoposide, Oxaliplatin or TRAIL individual treatments. # p<0.05 difference from control cells (Ctrl).
Mentions: We next investigated the role of Bid in the potential synergism between genotoxic drugs and TRAIL. Oxaliplatin and Etoposide both upregulated DR5 receptor expression in a time-dependent manner, while DR4 levels remained constant (Fig. 10 A, B). Similar to our data obtained with ER stressors, we observed potent synergistic effects of Oxaliplatin and Etoposide with TRAIL which was completely abolished in Bid-deficient cells (Fig. 10 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