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Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c.

Vaughn AE, Deshmukh M - Nat. Cell Biol. (2008)

Bottom Line: These two seemingly disparate cell types also show an increased regulation of the apoptotic pathway, which allows for their long-term survival.We report that the pro-apoptotic activity of cytochrome c is influenced by its redox state and that increases in reactive oxygen species (ROS) following an apoptotic insult lead to the oxidation and activation of cytochrome c.In healthy neurons and cancer cells, however, cytochrome c is reduced and held inactive by intracellular glutathione (GSH), generated as a result of glucose metabolism by the pentose phosphate pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Developmental Biology, Box 7250, 115 Mason Farm Road, Chapel Hill, North Carolina 27599, USA.

ABSTRACT
Neurons and cancer cells use glucose extensively, yet the precise advantage of this adaptation remains unclear. These two seemingly disparate cell types also show an increased regulation of the apoptotic pathway, which allows for their long-term survival. Here we show that both neurons and cancer cells strictly inhibit cytochrome c-mediated apoptosis by a mechanism dependent on glucose metabolism. We report that the pro-apoptotic activity of cytochrome c is influenced by its redox state and that increases in reactive oxygen species (ROS) following an apoptotic insult lead to the oxidation and activation of cytochrome c. In healthy neurons and cancer cells, however, cytochrome c is reduced and held inactive by intracellular glutathione (GSH), generated as a result of glucose metabolism by the pentose phosphate pathway. These results uncover a striking similarity in apoptosis regulation between neurons and cancer cells and provide insight into an adaptive advantage offered by the Warburg effect for cancer cell evasion of apoptosis and for long-term neuronal survival.

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Oxidation of cytochrome c increases its apoptotic activitya) XIAP -/- sympathetic neurons were treated with 20-50 μM of H2O2 for 20 minutes, followed by injection with cytochrome c protein and rhodamine, or rhodamine dye alone. Cell survival was assessed at various time points following injection. b) XIAP -/- sympathetic neurons were treated with 10 mM GSH ethyl ester for 12 hrs, followed by injection of cytochrome c along with rhodamine, or rhodamine alone. Cell survival was assessed at 16 hrs following injection. c) XIAP -/- sympathetic neurons were injected with either cytochrome c, or cytochrome c that was pre-incubated with 10 Units/ml of cytochrome c reductase. Cell survival was assessed at 16 hrs following injection. d) XIAP -/- sympathetic neurons were injected with either cytochrome c, or cytochrome c that had been pre-incubated with DTT (and subsequently separated) to reduce this cytochrome c. Percent survival was assessed after 6 hrs. e) XIAP -/- sympathetic neurons were deprived on NGF for 8 hrs in the presence of 10 mM GSH, followed by injection with EGFP or tBid-GFP constructs. Cell survival was quantified by cell morphology and expressed as a percentage of alive and healthy green cells at 16 hrs compared to 5 hrs post-injection. f) Exogenous cytochrome c (which is primarily oxidized) was added at a concentration of 10 uM to neuronal extracts, and Abs550 was measured following a 15 min. incubation. Control experiments measuring Abs550 of reduced or oxidized cytochrome c are also shown. Results are mean (±SEM) of three independent experiments.
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Figure 2: Oxidation of cytochrome c increases its apoptotic activitya) XIAP -/- sympathetic neurons were treated with 20-50 μM of H2O2 for 20 minutes, followed by injection with cytochrome c protein and rhodamine, or rhodamine dye alone. Cell survival was assessed at various time points following injection. b) XIAP -/- sympathetic neurons were treated with 10 mM GSH ethyl ester for 12 hrs, followed by injection of cytochrome c along with rhodamine, or rhodamine alone. Cell survival was assessed at 16 hrs following injection. c) XIAP -/- sympathetic neurons were injected with either cytochrome c, or cytochrome c that was pre-incubated with 10 Units/ml of cytochrome c reductase. Cell survival was assessed at 16 hrs following injection. d) XIAP -/- sympathetic neurons were injected with either cytochrome c, or cytochrome c that had been pre-incubated with DTT (and subsequently separated) to reduce this cytochrome c. Percent survival was assessed after 6 hrs. e) XIAP -/- sympathetic neurons were deprived on NGF for 8 hrs in the presence of 10 mM GSH, followed by injection with EGFP or tBid-GFP constructs. Cell survival was quantified by cell morphology and expressed as a percentage of alive and healthy green cells at 16 hrs compared to 5 hrs post-injection. f) Exogenous cytochrome c (which is primarily oxidized) was added at a concentration of 10 uM to neuronal extracts, and Abs550 was measured following a 15 min. incubation. Control experiments measuring Abs550 of reduced or oxidized cytochrome c are also shown. Results are mean (±SEM) of three independent experiments.

Mentions: To be apoptotically active, cytochrome c must exist as a holoenzyme complete with its heme prosthetic group12. In vitro studies have examined whether the redox state of cytochrome c affects its apoptotic activity. While some show that oxidized cytochrome c is more apoptotically active, others suggest that the reduced form can also function13-19. In particular, a recent study by Borutaite and Brown showed that oxidation of cytochrome c by cytochrome c oxidase promotes caspase activation, whereas its reduction by tertamethylphenylenediamine blocks caspase activation18. Likewise, oxidized but not reduced cytochrome c was found to promote apoptosis in permeabilized HepG2 cells19. To determine whether the intracellular redox environment affects the sensitivity of intact neurons to cytochrome c, we treated neurons with either low levels of hydrogen peroxide (H2O2) to create a more oxidized environment, or cell permeable reduced glutathione (GSH) to simulate a reduced environment. Neurons injected with 2.5 μg/μl cytochrome c exhibit substantial death only 10-16 hrs after the injections (Fig. 2a, b). However, H2O2 greatly increased the sensitivity of neurons to injected cytochrome c (Fig. 2a), while neurons treated with GSH were resistant (Fig. 2b). These results show that in intact cells, the redox environment has a dramatic affect on the ability of cytochrome c to promote apoptosis.


Glucose metabolism inhibits apoptosis in neurons and cancer cells by redox inactivation of cytochrome c.

Vaughn AE, Deshmukh M - Nat. Cell Biol. (2008)

Oxidation of cytochrome c increases its apoptotic activitya) XIAP -/- sympathetic neurons were treated with 20-50 μM of H2O2 for 20 minutes, followed by injection with cytochrome c protein and rhodamine, or rhodamine dye alone. Cell survival was assessed at various time points following injection. b) XIAP -/- sympathetic neurons were treated with 10 mM GSH ethyl ester for 12 hrs, followed by injection of cytochrome c along with rhodamine, or rhodamine alone. Cell survival was assessed at 16 hrs following injection. c) XIAP -/- sympathetic neurons were injected with either cytochrome c, or cytochrome c that was pre-incubated with 10 Units/ml of cytochrome c reductase. Cell survival was assessed at 16 hrs following injection. d) XIAP -/- sympathetic neurons were injected with either cytochrome c, or cytochrome c that had been pre-incubated with DTT (and subsequently separated) to reduce this cytochrome c. Percent survival was assessed after 6 hrs. e) XIAP -/- sympathetic neurons were deprived on NGF for 8 hrs in the presence of 10 mM GSH, followed by injection with EGFP or tBid-GFP constructs. Cell survival was quantified by cell morphology and expressed as a percentage of alive and healthy green cells at 16 hrs compared to 5 hrs post-injection. f) Exogenous cytochrome c (which is primarily oxidized) was added at a concentration of 10 uM to neuronal extracts, and Abs550 was measured following a 15 min. incubation. Control experiments measuring Abs550 of reduced or oxidized cytochrome c are also shown. Results are mean (±SEM) of three independent experiments.
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Related In: Results  -  Collection

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Figure 2: Oxidation of cytochrome c increases its apoptotic activitya) XIAP -/- sympathetic neurons were treated with 20-50 μM of H2O2 for 20 minutes, followed by injection with cytochrome c protein and rhodamine, or rhodamine dye alone. Cell survival was assessed at various time points following injection. b) XIAP -/- sympathetic neurons were treated with 10 mM GSH ethyl ester for 12 hrs, followed by injection of cytochrome c along with rhodamine, or rhodamine alone. Cell survival was assessed at 16 hrs following injection. c) XIAP -/- sympathetic neurons were injected with either cytochrome c, or cytochrome c that was pre-incubated with 10 Units/ml of cytochrome c reductase. Cell survival was assessed at 16 hrs following injection. d) XIAP -/- sympathetic neurons were injected with either cytochrome c, or cytochrome c that had been pre-incubated with DTT (and subsequently separated) to reduce this cytochrome c. Percent survival was assessed after 6 hrs. e) XIAP -/- sympathetic neurons were deprived on NGF for 8 hrs in the presence of 10 mM GSH, followed by injection with EGFP or tBid-GFP constructs. Cell survival was quantified by cell morphology and expressed as a percentage of alive and healthy green cells at 16 hrs compared to 5 hrs post-injection. f) Exogenous cytochrome c (which is primarily oxidized) was added at a concentration of 10 uM to neuronal extracts, and Abs550 was measured following a 15 min. incubation. Control experiments measuring Abs550 of reduced or oxidized cytochrome c are also shown. Results are mean (±SEM) of three independent experiments.
Mentions: To be apoptotically active, cytochrome c must exist as a holoenzyme complete with its heme prosthetic group12. In vitro studies have examined whether the redox state of cytochrome c affects its apoptotic activity. While some show that oxidized cytochrome c is more apoptotically active, others suggest that the reduced form can also function13-19. In particular, a recent study by Borutaite and Brown showed that oxidation of cytochrome c by cytochrome c oxidase promotes caspase activation, whereas its reduction by tertamethylphenylenediamine blocks caspase activation18. Likewise, oxidized but not reduced cytochrome c was found to promote apoptosis in permeabilized HepG2 cells19. To determine whether the intracellular redox environment affects the sensitivity of intact neurons to cytochrome c, we treated neurons with either low levels of hydrogen peroxide (H2O2) to create a more oxidized environment, or cell permeable reduced glutathione (GSH) to simulate a reduced environment. Neurons injected with 2.5 μg/μl cytochrome c exhibit substantial death only 10-16 hrs after the injections (Fig. 2a, b). However, H2O2 greatly increased the sensitivity of neurons to injected cytochrome c (Fig. 2a), while neurons treated with GSH were resistant (Fig. 2b). These results show that in intact cells, the redox environment has a dramatic affect on the ability of cytochrome c to promote apoptosis.

Bottom Line: These two seemingly disparate cell types also show an increased regulation of the apoptotic pathway, which allows for their long-term survival.We report that the pro-apoptotic activity of cytochrome c is influenced by its redox state and that increases in reactive oxygen species (ROS) following an apoptotic insult lead to the oxidation and activation of cytochrome c.In healthy neurons and cancer cells, however, cytochrome c is reduced and held inactive by intracellular glutathione (GSH), generated as a result of glucose metabolism by the pentose phosphate pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell & Developmental Biology, Box 7250, 115 Mason Farm Road, Chapel Hill, North Carolina 27599, USA.

ABSTRACT
Neurons and cancer cells use glucose extensively, yet the precise advantage of this adaptation remains unclear. These two seemingly disparate cell types also show an increased regulation of the apoptotic pathway, which allows for their long-term survival. Here we show that both neurons and cancer cells strictly inhibit cytochrome c-mediated apoptosis by a mechanism dependent on glucose metabolism. We report that the pro-apoptotic activity of cytochrome c is influenced by its redox state and that increases in reactive oxygen species (ROS) following an apoptotic insult lead to the oxidation and activation of cytochrome c. In healthy neurons and cancer cells, however, cytochrome c is reduced and held inactive by intracellular glutathione (GSH), generated as a result of glucose metabolism by the pentose phosphate pathway. These results uncover a striking similarity in apoptosis regulation between neurons and cancer cells and provide insight into an adaptive advantage offered by the Warburg effect for cancer cell evasion of apoptosis and for long-term neuronal survival.

Show MeSH
Related in: MedlinePlus