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Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome-c release.

Huber HJ, Dussmann H, Kilbride SM, Rehm M, Prehn JH - Mol. Syst. Biol. (2011)

Bottom Line: In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨ(m) from -142 to -88 mV, with active caspase-3 potentiating this decrease.ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨ(m).Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.

View Article: PubMed Central - PubMed

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

ABSTRACT
Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome-c (cyt-c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt-c release in these events. In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨ(m) from -142 to -88 mV, with active caspase-3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨ(m). However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt-c after release and (ii) the cell's glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.

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Model prediction: ATP synthase reversal stabilises ΔΨm via ATP consumption. (A) ATP synthase for the modelled cell in Figure 3 (A, B) is stably reversed from ∼10 min after the onset of cyt-c release in the presence (blue solid line) or absence (red dashed line) of caspase-3 feedback to complex I/II. (B) Inhibition of ATP synthase activity in the reversed mode according to the model in (A; and Figure 3) at time 30 min after onset of cyt-c release leads to a further depolarisation of ΔΨm, from −76 to −26 mV (model with caspase-3 cleavage of complex I/II) and from −88 to −65 mV (model without caspase-3 feedback to complex I/II). (C, D) Representative single-cell microscopic imaging of HeLa cells expressing cyt-c–GFP incubated with 30 nM TMRM and 100 μM caspase inhibitor zVAD-fmk 1 h prior to exposure to 3 μM staurosporine (STS). Five μM oligomycin was added as indicated. See text for further details on heterogeneous experimental outcome. (C) Fluorescent images of cyt-c–GFP release followed by TMRM depletion, which indicates depolarisation of ΔΨm. Residual TMRM staining is lost after addition of FCCP. Scale bar, 10 μm. (D) Traces of cyt-c release as indicated by the decrease in the s.d. of average GFP pixel intensity and mitochondrial depolarisation measured by the average pixel intensity of TMRM of two cells. Cell 1 is shown in (C). (E) Fractions of the cell population with reverse, forward or no ATP synthase activity detected by oligomycin responsiveness (237 cells, n=6 experiments). (F) ATP synthase proton flux before and after cyt-c release (t=10 min) according to the model in (A), with retention of 0.1% up to 2% respiration-accessible cyt-c. Calculations indicate a stable reversal of ATP synthase for <0.7% (blue regions) and ATP synthase in forward mode (light green to yellow) for more than 1.2% remaining respiration-accessible cyt-c. Source data is available for this figure at www.nature.com/msb.
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f4: Model prediction: ATP synthase reversal stabilises ΔΨm via ATP consumption. (A) ATP synthase for the modelled cell in Figure 3 (A, B) is stably reversed from ∼10 min after the onset of cyt-c release in the presence (blue solid line) or absence (red dashed line) of caspase-3 feedback to complex I/II. (B) Inhibition of ATP synthase activity in the reversed mode according to the model in (A; and Figure 3) at time 30 min after onset of cyt-c release leads to a further depolarisation of ΔΨm, from −76 to −26 mV (model with caspase-3 cleavage of complex I/II) and from −88 to −65 mV (model without caspase-3 feedback to complex I/II). (C, D) Representative single-cell microscopic imaging of HeLa cells expressing cyt-c–GFP incubated with 30 nM TMRM and 100 μM caspase inhibitor zVAD-fmk 1 h prior to exposure to 3 μM staurosporine (STS). Five μM oligomycin was added as indicated. See text for further details on heterogeneous experimental outcome. (C) Fluorescent images of cyt-c–GFP release followed by TMRM depletion, which indicates depolarisation of ΔΨm. Residual TMRM staining is lost after addition of FCCP. Scale bar, 10 μm. (D) Traces of cyt-c release as indicated by the decrease in the s.d. of average GFP pixel intensity and mitochondrial depolarisation measured by the average pixel intensity of TMRM of two cells. Cell 1 is shown in (C). (E) Fractions of the cell population with reverse, forward or no ATP synthase activity detected by oligomycin responsiveness (237 cells, n=6 experiments). (F) ATP synthase proton flux before and after cyt-c release (t=10 min) according to the model in (A), with retention of 0.1% up to 2% respiration-accessible cyt-c. Calculations indicate a stable reversal of ATP synthase for <0.7% (blue regions) and ATP synthase in forward mode (light green to yellow) for more than 1.2% remaining respiration-accessible cyt-c. Source data is available for this figure at www.nature.com/msb.

Mentions: Maintenance of ΔΨm subsequent to cyt-c release is essential for ionic homeostasis within the cell and thus may prevent the onset of necrotic cell death (Nicholls, 1977; Nicholls and Budd, 2000). Reversal of ATP synthase has been proposed to stabilise ΔΨm by pumping protons from the matrix into the IMS, while consuming instead of generating ATP (Goldstein et al, 2000; Chinopoulos and Adam-Vizi, 2009). However, it has also been reported that ΔΨm can stabilise in the absence of ATP synthase reversal. We therefore aimed to investigate whether and under what conditions ATP synthase reversal was present and if so what the implications on cytosolic ATP and ΔΨm were. We first confirmed that ATP synthase activity was reversed 10 min after onset of cyt-c release when respiration-accessible cyt-c decreased to 0.1% in our model (Figure 4A, blue solid line). This was slightly more pronounced when caspase-3 deactivation of complex I/II was also taken into consideration (Figure 4A, red dashed line). Moreover, our model confirmed that ATP synthase reversal consumed ATP (Supplementary Figure 6A), that glycolysis prevented ATP depletion during this reversal (Supplementary Figure 6B) and that ATP synthase reversal was robust when model parameters (Supplementary Table VII) were increased or decreased over a fivefold parameter range (Supplementary Figure 7A and Supplementary Text II).


Glucose metabolism determines resistance of cancer cells to bioenergetic crisis after cytochrome-c release.

Huber HJ, Dussmann H, Kilbride SM, Rehm M, Prehn JH - Mol. Syst. Biol. (2011)

Model prediction: ATP synthase reversal stabilises ΔΨm via ATP consumption. (A) ATP synthase for the modelled cell in Figure 3 (A, B) is stably reversed from ∼10 min after the onset of cyt-c release in the presence (blue solid line) or absence (red dashed line) of caspase-3 feedback to complex I/II. (B) Inhibition of ATP synthase activity in the reversed mode according to the model in (A; and Figure 3) at time 30 min after onset of cyt-c release leads to a further depolarisation of ΔΨm, from −76 to −26 mV (model with caspase-3 cleavage of complex I/II) and from −88 to −65 mV (model without caspase-3 feedback to complex I/II). (C, D) Representative single-cell microscopic imaging of HeLa cells expressing cyt-c–GFP incubated with 30 nM TMRM and 100 μM caspase inhibitor zVAD-fmk 1 h prior to exposure to 3 μM staurosporine (STS). Five μM oligomycin was added as indicated. See text for further details on heterogeneous experimental outcome. (C) Fluorescent images of cyt-c–GFP release followed by TMRM depletion, which indicates depolarisation of ΔΨm. Residual TMRM staining is lost after addition of FCCP. Scale bar, 10 μm. (D) Traces of cyt-c release as indicated by the decrease in the s.d. of average GFP pixel intensity and mitochondrial depolarisation measured by the average pixel intensity of TMRM of two cells. Cell 1 is shown in (C). (E) Fractions of the cell population with reverse, forward or no ATP synthase activity detected by oligomycin responsiveness (237 cells, n=6 experiments). (F) ATP synthase proton flux before and after cyt-c release (t=10 min) according to the model in (A), with retention of 0.1% up to 2% respiration-accessible cyt-c. Calculations indicate a stable reversal of ATP synthase for <0.7% (blue regions) and ATP synthase in forward mode (light green to yellow) for more than 1.2% remaining respiration-accessible cyt-c. Source data is available for this figure at www.nature.com/msb.
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Related In: Results  -  Collection

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f4: Model prediction: ATP synthase reversal stabilises ΔΨm via ATP consumption. (A) ATP synthase for the modelled cell in Figure 3 (A, B) is stably reversed from ∼10 min after the onset of cyt-c release in the presence (blue solid line) or absence (red dashed line) of caspase-3 feedback to complex I/II. (B) Inhibition of ATP synthase activity in the reversed mode according to the model in (A; and Figure 3) at time 30 min after onset of cyt-c release leads to a further depolarisation of ΔΨm, from −76 to −26 mV (model with caspase-3 cleavage of complex I/II) and from −88 to −65 mV (model without caspase-3 feedback to complex I/II). (C, D) Representative single-cell microscopic imaging of HeLa cells expressing cyt-c–GFP incubated with 30 nM TMRM and 100 μM caspase inhibitor zVAD-fmk 1 h prior to exposure to 3 μM staurosporine (STS). Five μM oligomycin was added as indicated. See text for further details on heterogeneous experimental outcome. (C) Fluorescent images of cyt-c–GFP release followed by TMRM depletion, which indicates depolarisation of ΔΨm. Residual TMRM staining is lost after addition of FCCP. Scale bar, 10 μm. (D) Traces of cyt-c release as indicated by the decrease in the s.d. of average GFP pixel intensity and mitochondrial depolarisation measured by the average pixel intensity of TMRM of two cells. Cell 1 is shown in (C). (E) Fractions of the cell population with reverse, forward or no ATP synthase activity detected by oligomycin responsiveness (237 cells, n=6 experiments). (F) ATP synthase proton flux before and after cyt-c release (t=10 min) according to the model in (A), with retention of 0.1% up to 2% respiration-accessible cyt-c. Calculations indicate a stable reversal of ATP synthase for <0.7% (blue regions) and ATP synthase in forward mode (light green to yellow) for more than 1.2% remaining respiration-accessible cyt-c. Source data is available for this figure at www.nature.com/msb.
Mentions: Maintenance of ΔΨm subsequent to cyt-c release is essential for ionic homeostasis within the cell and thus may prevent the onset of necrotic cell death (Nicholls, 1977; Nicholls and Budd, 2000). Reversal of ATP synthase has been proposed to stabilise ΔΨm by pumping protons from the matrix into the IMS, while consuming instead of generating ATP (Goldstein et al, 2000; Chinopoulos and Adam-Vizi, 2009). However, it has also been reported that ΔΨm can stabilise in the absence of ATP synthase reversal. We therefore aimed to investigate whether and under what conditions ATP synthase reversal was present and if so what the implications on cytosolic ATP and ΔΨm were. We first confirmed that ATP synthase activity was reversed 10 min after onset of cyt-c release when respiration-accessible cyt-c decreased to 0.1% in our model (Figure 4A, blue solid line). This was slightly more pronounced when caspase-3 deactivation of complex I/II was also taken into consideration (Figure 4A, red dashed line). Moreover, our model confirmed that ATP synthase reversal consumed ATP (Supplementary Figure 6A), that glycolysis prevented ATP depletion during this reversal (Supplementary Figure 6B) and that ATP synthase reversal was robust when model parameters (Supplementary Table VII) were increased or decreased over a fivefold parameter range (Supplementary Figure 7A and Supplementary Text II).

Bottom Line: In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨ(m) from -142 to -88 mV, with active caspase-3 potentiating this decrease.ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨ(m).Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.

View Article: PubMed Central - PubMed

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

ABSTRACT
Many anticancer drugs activate caspases via the mitochondrial apoptosis pathway. Activation of this pathway triggers a concomitant bioenergetic crisis caused by the release of cytochrome-c (cyt-c). Cancer cells are able to evade these processes by altering metabolic and caspase activation pathways. In this study, we provide the first integrated system study of mitochondrial bioenergetics and apoptosis signalling and examine the role of mitochondrial cyt-c release in these events. In accordance with single-cell experiments, our model showed that loss of cyt-c decreased mitochondrial respiration by 95% and depolarised mitochondrial membrane potential ΔΨ(m) from -142 to -88 mV, with active caspase-3 potentiating this decrease. ATP synthase was reversed under such conditions, consuming ATP and stabilising ΔΨ(m). However, the direction and level of ATP synthase activity showed significant heterogeneity in individual cancer cells, which the model explained by variations in (i) accessible cyt-c after release and (ii) the cell's glycolytic capacity. Our results provide a quantitative and mechanistic explanation for the protective role of enhanced glucose utilisation for cancer cells to avert the otherwise lethal bioenergetic crisis associated with apoptosis initiation.

Show MeSH
Related in: MedlinePlus