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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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Glucose metabolism protects cancer cells from cytochrome c-mediated apoptosisa) Normal cells (Human Mammary Epithelial Cells-HuMECs, MEFs, Human Dermal Fibroblasts-HDFs) or cancer cell lines (Sk-Mel 103, HeLa, JM2) were injected with cytochrome c and Smac protein and cell survival was assessed after 30 minutes. b) Total glutathione (GSH) was measured in normal mitotic cells as well as cancer cell lines, and expressed as a concentration of GSH to total cellular protein. c) MEFs were treated with 5 mM GSH ethyl ester for 15 min, followed by injection with cytochrome c. After 1 hr, injected cells were assessed for Annexin V positivity. Photographs show representative Annexin V-FITC staining of cytochrome c injected cells (arrows). d) Exogenous cytochrome c (which is primarily oxidized) was added at a concentration of 10 μM to normal or cancer cell extract, and Abs550 was measured following a 15 min incubation. e) Average ROS levels in HeLa cells measured by fluorescence of CMH2DCFDA in the absence or presence of the pentose phosphate pathway inhibitor, DHEA (200 μM) for 6 hrs. f) The pentose phosphate pathway was inhibited in various cancer cell lines for 6 hrs by addition of 200 μM DHEA, followed by injection of cytochrome c and Smac protein. Cell survival was assessed at 3 hrs. g) JM2 and HeLa cells were deprived of glucose for 16 hrs followed by injection with cytochrome c and Smac, and assessed for survival at various time points. Images are representative of JM2 cells at 3 hrs following cytochrome c injection. Error bars represent ±SEM of n≥3.
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Figure 4: Glucose metabolism protects cancer cells from cytochrome c-mediated apoptosisa) Normal cells (Human Mammary Epithelial Cells-HuMECs, MEFs, Human Dermal Fibroblasts-HDFs) or cancer cell lines (Sk-Mel 103, HeLa, JM2) were injected with cytochrome c and Smac protein and cell survival was assessed after 30 minutes. b) Total glutathione (GSH) was measured in normal mitotic cells as well as cancer cell lines, and expressed as a concentration of GSH to total cellular protein. c) MEFs were treated with 5 mM GSH ethyl ester for 15 min, followed by injection with cytochrome c. After 1 hr, injected cells were assessed for Annexin V positivity. Photographs show representative Annexin V-FITC staining of cytochrome c injected cells (arrows). d) Exogenous cytochrome c (which is primarily oxidized) was added at a concentration of 10 μM to normal or cancer cell extract, and Abs550 was measured following a 15 min incubation. e) Average ROS levels in HeLa cells measured by fluorescence of CMH2DCFDA in the absence or presence of the pentose phosphate pathway inhibitor, DHEA (200 μM) for 6 hrs. f) The pentose phosphate pathway was inhibited in various cancer cell lines for 6 hrs by addition of 200 μM DHEA, followed by injection of cytochrome c and Smac protein. Cell survival was assessed at 3 hrs. g) JM2 and HeLa cells were deprived of glucose for 16 hrs followed by injection with cytochrome c and Smac, and assessed for survival at various time points. Images are representative of JM2 cells at 3 hrs following cytochrome c injection. Error bars represent ±SEM of n≥3.

Mentions: Like neurons, many cancer cells are known to metabolize glucose extensively, a phenomenon known as the Warburg effect6. To investigate whether increased glucose metabolism in cancer cells directly regulates apoptosis at the point of cytochrome c, we first examined the sensitivity of cancer cells to cytosolic injection of cytochrome c. Cytochrome c was injected in the presence of Smac to eliminate the activity of IAPs that are known to block caspase activation in many cancer cells25, thus focusing on IAP-independent mechanisms of cytochrome c regulation. While normal cells readily underwent apoptosis in response to cytosolic cytochrome c, cancer cells showed an increased resistance (Fig. 4a, Supplementary Information, Fig. S10a). Strikingly, the decreased sensitivity to cytochrome c correlated well with the increased levels of intracellular GSH found in these cancer cells (Fig. 4b). Interestingly, treatment of MEFs with cell permeable GSH increased their resistance to cytosolic cytochrome c (Fig. 4c). In addition, while incubation of oxidized cytochrome c with MEF lysate did not significantly affect the redox state of oxidized cytochrome c, oxidized cytochrome c was rapidly reduced upon incubation with lysates generated from cancer cells (Fig. 4d).


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

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

Glucose metabolism protects cancer cells from cytochrome c-mediated apoptosisa) Normal cells (Human Mammary Epithelial Cells-HuMECs, MEFs, Human Dermal Fibroblasts-HDFs) or cancer cell lines (Sk-Mel 103, HeLa, JM2) were injected with cytochrome c and Smac protein and cell survival was assessed after 30 minutes. b) Total glutathione (GSH) was measured in normal mitotic cells as well as cancer cell lines, and expressed as a concentration of GSH to total cellular protein. c) MEFs were treated with 5 mM GSH ethyl ester for 15 min, followed by injection with cytochrome c. After 1 hr, injected cells were assessed for Annexin V positivity. Photographs show representative Annexin V-FITC staining of cytochrome c injected cells (arrows). d) Exogenous cytochrome c (which is primarily oxidized) was added at a concentration of 10 μM to normal or cancer cell extract, and Abs550 was measured following a 15 min incubation. e) Average ROS levels in HeLa cells measured by fluorescence of CMH2DCFDA in the absence or presence of the pentose phosphate pathway inhibitor, DHEA (200 μM) for 6 hrs. f) The pentose phosphate pathway was inhibited in various cancer cell lines for 6 hrs by addition of 200 μM DHEA, followed by injection of cytochrome c and Smac protein. Cell survival was assessed at 3 hrs. g) JM2 and HeLa cells were deprived of glucose for 16 hrs followed by injection with cytochrome c and Smac, and assessed for survival at various time points. Images are representative of JM2 cells at 3 hrs following cytochrome c injection. Error bars represent ±SEM of n≥3.
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Figure 4: Glucose metabolism protects cancer cells from cytochrome c-mediated apoptosisa) Normal cells (Human Mammary Epithelial Cells-HuMECs, MEFs, Human Dermal Fibroblasts-HDFs) or cancer cell lines (Sk-Mel 103, HeLa, JM2) were injected with cytochrome c and Smac protein and cell survival was assessed after 30 minutes. b) Total glutathione (GSH) was measured in normal mitotic cells as well as cancer cell lines, and expressed as a concentration of GSH to total cellular protein. c) MEFs were treated with 5 mM GSH ethyl ester for 15 min, followed by injection with cytochrome c. After 1 hr, injected cells were assessed for Annexin V positivity. Photographs show representative Annexin V-FITC staining of cytochrome c injected cells (arrows). d) Exogenous cytochrome c (which is primarily oxidized) was added at a concentration of 10 μM to normal or cancer cell extract, and Abs550 was measured following a 15 min incubation. e) Average ROS levels in HeLa cells measured by fluorescence of CMH2DCFDA in the absence or presence of the pentose phosphate pathway inhibitor, DHEA (200 μM) for 6 hrs. f) The pentose phosphate pathway was inhibited in various cancer cell lines for 6 hrs by addition of 200 μM DHEA, followed by injection of cytochrome c and Smac protein. Cell survival was assessed at 3 hrs. g) JM2 and HeLa cells were deprived of glucose for 16 hrs followed by injection with cytochrome c and Smac, and assessed for survival at various time points. Images are representative of JM2 cells at 3 hrs following cytochrome c injection. Error bars represent ±SEM of n≥3.
Mentions: Like neurons, many cancer cells are known to metabolize glucose extensively, a phenomenon known as the Warburg effect6. To investigate whether increased glucose metabolism in cancer cells directly regulates apoptosis at the point of cytochrome c, we first examined the sensitivity of cancer cells to cytosolic injection of cytochrome c. Cytochrome c was injected in the presence of Smac to eliminate the activity of IAPs that are known to block caspase activation in many cancer cells25, thus focusing on IAP-independent mechanisms of cytochrome c regulation. While normal cells readily underwent apoptosis in response to cytosolic cytochrome c, cancer cells showed an increased resistance (Fig. 4a, Supplementary Information, Fig. S10a). Strikingly, the decreased sensitivity to cytochrome c correlated well with the increased levels of intracellular GSH found in these cancer cells (Fig. 4b). Interestingly, treatment of MEFs with cell permeable GSH increased their resistance to cytosolic cytochrome c (Fig. 4c). In addition, while incubation of oxidized cytochrome c with MEF lysate did not significantly affect the redox state of oxidized cytochrome c, oxidized cytochrome c was rapidly reduced upon incubation with lysates generated from cancer cells (Fig. 4d).

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