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D-2-Hydroxyglutarate does not mimic all the IDH mutation effects, in particular the reduced etoposide-triggered apoptosis mediated by an alteration in mitochondrial NADH.

Oizel K, Gratas C, Nadaradjane A, Oliver L, Vallette FM, Pecqueur C - Cell Death Dis (2015)

Bottom Line: The present study is aimed at deciphering how the mutant IDH can affect cancer pathogenesis, in particular with respect to its associated oncometabolite D-2HG.However, although mutant IDH reduced cell sensitivity to the apoptotic inducer etoposide, D-2HG exhibited no effect on apoptosis.Instead, we found that the apoptotic effect was mediated through the mitochondrial NADH pool reduction and could be inhibited by oxamate.

View Article: PubMed Central - PubMed

Affiliation: 1] CRCNA - INSERM UMR 892 - CNRS UMR 6299, Nantes F44007, France [2] Faculté de Médecine, Université de Nantes, Nantes F44007, France.

ABSTRACT
Somatic mutations in isocitrate dehydrogenase (IDH)-1 and -2 have recently been described in glioma. This mutation leads to a neomorphic enzymatic activity as the conversion of isocitrate to alpha ketoglutarate (αKG) is replaced by the conversion of αKG to D-2-hydroxyglutarate (D-2HG) with NADPH oxidation. It has been suggested that this oncometabolite D-2HG via inhibition of αKG-dioxygenases is involved in multiple functions such as epigenetic modifications or hypoxia responses. The present study is aimed at deciphering how the mutant IDH can affect cancer pathogenesis, in particular with respect to its associated oncometabolite D-2HG. We show that the overexpression of mutant IDH in glioma cells or treatment with D-2HG triggered an increase in cell proliferation. However, although mutant IDH reduced cell sensitivity to the apoptotic inducer etoposide, D-2HG exhibited no effect on apoptosis. Instead, we found that the apoptotic effect was mediated through the mitochondrial NADH pool reduction and could be inhibited by oxamate. These data show that besides D-2HG production, mutant IDH affects other crucial metabolite pools. These observations lead to a better understanding of the biology of IDH mutations in gliomas and their response to therapy.

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Related in: MedlinePlus

(a) Expression of proteins involved in apoptosis in U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms. Whole lysates of cells were isolated 24 h after vehicule (V) or etoposide (ETO) treatment and analyzed (40 μg) by immunoblotting with the indicated antibodies (left panel). Actin was used as a loading control. (b) ROS production was measured using the DCFDA probe. U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms were seeded at 2.5 × 104 cells then incubated with the DCFDA probe. Fluorescence was measured at 538 nm every 3 min for 75 min and the slope corresponding to ROS production was calculated. (c) Oxygen consumption rate (OCR) of stable cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured over time. Cells (4 × 104) were plated and OCR was measured 24 h later by a XF24 Analyzer (Seahorse Bioscience). Mitochondrial inhibitors (oligomycin(a), CCCP(b), and rotenone and antimycin A(c)) were added as indicated with arrows. (d) Basal oxygen consumption was determined by measuring OCR as in c removing the non-mitochondrial oxygen consumption (OCR upon rotenone and antimycin A treatment). (e) The respiratory reserve of U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured by a XF24 Analyzer (Seahorse Bioscience). The respiratory reserve was determined as the difference between maximal OCR and basal OCR. (f) NADH production of cells expressing wild-type or mutant IDH1 isoforms. Cells (1 × 106) were plated, lysed the next day and subsequently assayed for their ability to produce NADH. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate
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fig4: (a) Expression of proteins involved in apoptosis in U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms. Whole lysates of cells were isolated 24 h after vehicule (V) or etoposide (ETO) treatment and analyzed (40 μg) by immunoblotting with the indicated antibodies (left panel). Actin was used as a loading control. (b) ROS production was measured using the DCFDA probe. U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms were seeded at 2.5 × 104 cells then incubated with the DCFDA probe. Fluorescence was measured at 538 nm every 3 min for 75 min and the slope corresponding to ROS production was calculated. (c) Oxygen consumption rate (OCR) of stable cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured over time. Cells (4 × 104) were plated and OCR was measured 24 h later by a XF24 Analyzer (Seahorse Bioscience). Mitochondrial inhibitors (oligomycin(a), CCCP(b), and rotenone and antimycin A(c)) were added as indicated with arrows. (d) Basal oxygen consumption was determined by measuring OCR as in c removing the non-mitochondrial oxygen consumption (OCR upon rotenone and antimycin A treatment). (e) The respiratory reserve of U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured by a XF24 Analyzer (Seahorse Bioscience). The respiratory reserve was determined as the difference between maximal OCR and basal OCR. (f) NADH production of cells expressing wild-type or mutant IDH1 isoforms. Cells (1 × 106) were plated, lysed the next day and subsequently assayed for their ability to produce NADH. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate

Mentions: To better understand how IDH1R132 affects apoptosis, several parameters were analyzed. IDH mutation can alter protein expression through epigenetic modifications, redox homeostasis and metabolism of glucose, glutamine (Gln) and fatty acids. As an aberrant methylation status of Bax and Bcl2 has been shown to be associated with apoptosis escape,15 expression of these proteins, as well as BclXL, XIAP, truncated BID (tBID) and survivin, was analyzed by western blot (Figure 4a). However, neither IDH1R132 nor wild-type IDH1 expression affected protein expression of either control or ETO-treated cells. Expression of Bax and Bcl2 increased upon ETO treatment but to the same extent in vector, IDH1- and IDH1R132-expressing cells. Of note, a similar pattern of expression of Bax and Bcl2 was observed in cells treated with αKG or D-2HG before ETO (Supplementary Figure S1). As ROS and mitochondria have an important role in apoptosis, we then measured ROS production using the DCFDA probe and cell metabolism using the XF24 analyzer. Cellular ROS were not affected by either wild-type or IDH1R132 overexpression as shown in Figure 4b. Of note, ROS production was increased with rotenone, an inhibitor of the mitochondrial complex I able to induce mitochondrial ROS production, and decreased with EGCG, a polyphenol compound known for its antioxidant effect (Supplementary Figure S2). Interestingly, overexpression of IDH1R132 and IDH2R172 was associated with a slight but significant decreased in mitochondrial oxygen consumption rate (OCR) (Figures 4c and d, Supplementary Figure S3A). In addition to the basal OCR, maximal consumption rate, spare capacity and coupling efficiency were calculated from the recordings of the OCR following addition of different mitochondrial inhibitors (Figure 4c). Oligomycin is an inhibitor of ATP synthase and can be used to determine mitochondrial coupling efficiency, the efficiency with which mitochondria convert oxygen into ATP. CCCP, an uncoupler of mitochondrial oxidative phosphorylation raises OCR to its maximal rate, which allows the calculation of the respiratory reserve. Finally, rotenone and antimycin A, respectively, inhibits the complex I and complex III, which allows the determination of the non-mitochondrial oxygen consumption. Interestingly, a reduced respiratory reserve was observed in cells overexpressing IDH1R132 (Figure 4e) or IDH2R172 (Supplementary Figure S3B). To determine which complex was involved in the decreased respiratory reserve, OCR was recorded after sequential addition of rotenone and antimycin A. Complex I activity (left panel) contributed for 90% of total OCR, whereas complex II (right panel) contributed for only 10% (Supplementary Figure S4). Mutant IDH did not change the respective contribution of these complexes to mitochondrial respiration. Taken into account that the majority of the electrons entering the electron transport chain (ETC) are doing so at the level of complex I through oxidation of NADH, the decreased mitochondrial respiratory reserve triggered by mutant IDH reflects probably a reduction of the NADH mitochondrial pool. In order to confirm this hypothesis, NADH level was measured in our cells (Figure 4f). Indeed, NADH level was decreased in IDH1R132 cells compared with IDH1 cells.


D-2-Hydroxyglutarate does not mimic all the IDH mutation effects, in particular the reduced etoposide-triggered apoptosis mediated by an alteration in mitochondrial NADH.

Oizel K, Gratas C, Nadaradjane A, Oliver L, Vallette FM, Pecqueur C - Cell Death Dis (2015)

(a) Expression of proteins involved in apoptosis in U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms. Whole lysates of cells were isolated 24 h after vehicule (V) or etoposide (ETO) treatment and analyzed (40 μg) by immunoblotting with the indicated antibodies (left panel). Actin was used as a loading control. (b) ROS production was measured using the DCFDA probe. U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms were seeded at 2.5 × 104 cells then incubated with the DCFDA probe. Fluorescence was measured at 538 nm every 3 min for 75 min and the slope corresponding to ROS production was calculated. (c) Oxygen consumption rate (OCR) of stable cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured over time. Cells (4 × 104) were plated and OCR was measured 24 h later by a XF24 Analyzer (Seahorse Bioscience). Mitochondrial inhibitors (oligomycin(a), CCCP(b), and rotenone and antimycin A(c)) were added as indicated with arrows. (d) Basal oxygen consumption was determined by measuring OCR as in c removing the non-mitochondrial oxygen consumption (OCR upon rotenone and antimycin A treatment). (e) The respiratory reserve of U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured by a XF24 Analyzer (Seahorse Bioscience). The respiratory reserve was determined as the difference between maximal OCR and basal OCR. (f) NADH production of cells expressing wild-type or mutant IDH1 isoforms. Cells (1 × 106) were plated, lysed the next day and subsequently assayed for their ability to produce NADH. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4385911&req=5

fig4: (a) Expression of proteins involved in apoptosis in U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms. Whole lysates of cells were isolated 24 h after vehicule (V) or etoposide (ETO) treatment and analyzed (40 μg) by immunoblotting with the indicated antibodies (left panel). Actin was used as a loading control. (b) ROS production was measured using the DCFDA probe. U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms were seeded at 2.5 × 104 cells then incubated with the DCFDA probe. Fluorescence was measured at 538 nm every 3 min for 75 min and the slope corresponding to ROS production was calculated. (c) Oxygen consumption rate (OCR) of stable cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured over time. Cells (4 × 104) were plated and OCR was measured 24 h later by a XF24 Analyzer (Seahorse Bioscience). Mitochondrial inhibitors (oligomycin(a), CCCP(b), and rotenone and antimycin A(c)) were added as indicated with arrows. (d) Basal oxygen consumption was determined by measuring OCR as in c removing the non-mitochondrial oxygen consumption (OCR upon rotenone and antimycin A treatment). (e) The respiratory reserve of U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms was measured by a XF24 Analyzer (Seahorse Bioscience). The respiratory reserve was determined as the difference between maximal OCR and basal OCR. (f) NADH production of cells expressing wild-type or mutant IDH1 isoforms. Cells (1 × 106) were plated, lysed the next day and subsequently assayed for their ability to produce NADH. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate
Mentions: To better understand how IDH1R132 affects apoptosis, several parameters were analyzed. IDH mutation can alter protein expression through epigenetic modifications, redox homeostasis and metabolism of glucose, glutamine (Gln) and fatty acids. As an aberrant methylation status of Bax and Bcl2 has been shown to be associated with apoptosis escape,15 expression of these proteins, as well as BclXL, XIAP, truncated BID (tBID) and survivin, was analyzed by western blot (Figure 4a). However, neither IDH1R132 nor wild-type IDH1 expression affected protein expression of either control or ETO-treated cells. Expression of Bax and Bcl2 increased upon ETO treatment but to the same extent in vector, IDH1- and IDH1R132-expressing cells. Of note, a similar pattern of expression of Bax and Bcl2 was observed in cells treated with αKG or D-2HG before ETO (Supplementary Figure S1). As ROS and mitochondria have an important role in apoptosis, we then measured ROS production using the DCFDA probe and cell metabolism using the XF24 analyzer. Cellular ROS were not affected by either wild-type or IDH1R132 overexpression as shown in Figure 4b. Of note, ROS production was increased with rotenone, an inhibitor of the mitochondrial complex I able to induce mitochondrial ROS production, and decreased with EGCG, a polyphenol compound known for its antioxidant effect (Supplementary Figure S2). Interestingly, overexpression of IDH1R132 and IDH2R172 was associated with a slight but significant decreased in mitochondrial oxygen consumption rate (OCR) (Figures 4c and d, Supplementary Figure S3A). In addition to the basal OCR, maximal consumption rate, spare capacity and coupling efficiency were calculated from the recordings of the OCR following addition of different mitochondrial inhibitors (Figure 4c). Oligomycin is an inhibitor of ATP synthase and can be used to determine mitochondrial coupling efficiency, the efficiency with which mitochondria convert oxygen into ATP. CCCP, an uncoupler of mitochondrial oxidative phosphorylation raises OCR to its maximal rate, which allows the calculation of the respiratory reserve. Finally, rotenone and antimycin A, respectively, inhibits the complex I and complex III, which allows the determination of the non-mitochondrial oxygen consumption. Interestingly, a reduced respiratory reserve was observed in cells overexpressing IDH1R132 (Figure 4e) or IDH2R172 (Supplementary Figure S3B). To determine which complex was involved in the decreased respiratory reserve, OCR was recorded after sequential addition of rotenone and antimycin A. Complex I activity (left panel) contributed for 90% of total OCR, whereas complex II (right panel) contributed for only 10% (Supplementary Figure S4). Mutant IDH did not change the respective contribution of these complexes to mitochondrial respiration. Taken into account that the majority of the electrons entering the electron transport chain (ETC) are doing so at the level of complex I through oxidation of NADH, the decreased mitochondrial respiratory reserve triggered by mutant IDH reflects probably a reduction of the NADH mitochondrial pool. In order to confirm this hypothesis, NADH level was measured in our cells (Figure 4f). Indeed, NADH level was decreased in IDH1R132 cells compared with IDH1 cells.

Bottom Line: The present study is aimed at deciphering how the mutant IDH can affect cancer pathogenesis, in particular with respect to its associated oncometabolite D-2HG.However, although mutant IDH reduced cell sensitivity to the apoptotic inducer etoposide, D-2HG exhibited no effect on apoptosis.Instead, we found that the apoptotic effect was mediated through the mitochondrial NADH pool reduction and could be inhibited by oxamate.

View Article: PubMed Central - PubMed

Affiliation: 1] CRCNA - INSERM UMR 892 - CNRS UMR 6299, Nantes F44007, France [2] Faculté de Médecine, Université de Nantes, Nantes F44007, France.

ABSTRACT
Somatic mutations in isocitrate dehydrogenase (IDH)-1 and -2 have recently been described in glioma. This mutation leads to a neomorphic enzymatic activity as the conversion of isocitrate to alpha ketoglutarate (αKG) is replaced by the conversion of αKG to D-2-hydroxyglutarate (D-2HG) with NADPH oxidation. It has been suggested that this oncometabolite D-2HG via inhibition of αKG-dioxygenases is involved in multiple functions such as epigenetic modifications or hypoxia responses. The present study is aimed at deciphering how the mutant IDH can affect cancer pathogenesis, in particular with respect to its associated oncometabolite D-2HG. We show that the overexpression of mutant IDH in glioma cells or treatment with D-2HG triggered an increase in cell proliferation. However, although mutant IDH reduced cell sensitivity to the apoptotic inducer etoposide, D-2HG exhibited no effect on apoptosis. Instead, we found that the apoptotic effect was mediated through the mitochondrial NADH pool reduction and could be inhibited by oxamate. These data show that besides D-2HG production, mutant IDH affects other crucial metabolite pools. These observations lead to a better understanding of the biology of IDH mutations in gliomas and their response to therapy.

Show MeSH
Related in: MedlinePlus