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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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(a) Expression of wild-type (IDH2) or mutant IDH2 (R140 and R172) isoforms in stable overexpressing U251 cells was confirmed by immunoblotting using whole lysates (40 μg) or purified mitochondria. Actin and porin were used as a loading control, respectively, for whole lysates and mitochondria. (b) IDH activity in cells overexpressing wild-type or mutant IDH2 isoforms. Cells (1 × 106) were plated, lysed the next day and subsequently assayed for their ability to generate NADPH. (c) Caspase 3 activation was determined with DEVDase activity assay in stable U251 cells expressing wild-type and mutant IDH2 isoforms after 24 h ETO (50 μg/ml) exposure. Results are expressed relative to wild-type IDH2-expressing cells. (d) Caspase 3 activation was determined with DEVDase activity assay after 24 h ETO (50 μg/ml) exposure in U251 cells treated with D-2HG (3 mM) for 6 days. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH2, wild-type IDH2-expressing cells; R140, IDH1R140-expressing cells transfected; R172, IDH1R172-expressing cells transfected. *P<0.05, **P<0.01 and ***P<0.001
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fig3: (a) Expression of wild-type (IDH2) or mutant IDH2 (R140 and R172) isoforms in stable overexpressing U251 cells was confirmed by immunoblotting using whole lysates (40 μg) or purified mitochondria. Actin and porin were used as a loading control, respectively, for whole lysates and mitochondria. (b) IDH activity in cells overexpressing wild-type or mutant IDH2 isoforms. Cells (1 × 106) were plated, lysed the next day and subsequently assayed for their ability to generate NADPH. (c) Caspase 3 activation was determined with DEVDase activity assay in stable U251 cells expressing wild-type and mutant IDH2 isoforms after 24 h ETO (50 μg/ml) exposure. Results are expressed relative to wild-type IDH2-expressing cells. (d) Caspase 3 activation was determined with DEVDase activity assay after 24 h ETO (50 μg/ml) exposure in U251 cells treated with D-2HG (3 mM) for 6 days. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH2, wild-type IDH2-expressing cells; R140, IDH1R140-expressing cells transfected; R172, IDH1R172-expressing cells transfected. *P<0.05, **P<0.01 and ***P<0.001

Mentions: To determine whether mutant IDH2 triggered similar effects on cell death, U251 cells were transfected with vector, IDH2, IDH2R140 and IDH2R172. Western blot analysis showed that IDH2 proteins were expressed in the mitochondria and not in the cytosol (Figure 3a). Similarly to IDH1R132, mutant IDH2-expressing cells exhibited decreased NADPH consumption corresponding to its neomorphic activity (Figure 3b). Interestingly, caspase-3 activation following ETO treatment was also significantly reduced (Figure 3c). Altogether, these results showed that all mutants IDH, independently of their respective subcellular localization, reduced ETO-induced cell death.


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 wild-type (IDH2) or mutant IDH2 (R140 and R172) isoforms in stable overexpressing U251 cells was confirmed by immunoblotting using whole lysates (40 μg) or purified mitochondria. Actin and porin were used as a loading control, respectively, for whole lysates and mitochondria. (b) IDH activity in cells overexpressing wild-type or mutant IDH2 isoforms. Cells (1 × 106) were plated, lysed the next day and subsequently assayed for their ability to generate NADPH. (c) Caspase 3 activation was determined with DEVDase activity assay in stable U251 cells expressing wild-type and mutant IDH2 isoforms after 24 h ETO (50 μg/ml) exposure. Results are expressed relative to wild-type IDH2-expressing cells. (d) Caspase 3 activation was determined with DEVDase activity assay after 24 h ETO (50 μg/ml) exposure in U251 cells treated with D-2HG (3 mM) for 6 days. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH2, wild-type IDH2-expressing cells; R140, IDH1R140-expressing cells transfected; R172, IDH1R172-expressing cells transfected. *P<0.05, **P<0.01 and ***P<0.001
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig3: (a) Expression of wild-type (IDH2) or mutant IDH2 (R140 and R172) isoforms in stable overexpressing U251 cells was confirmed by immunoblotting using whole lysates (40 μg) or purified mitochondria. Actin and porin were used as a loading control, respectively, for whole lysates and mitochondria. (b) IDH activity in cells overexpressing wild-type or mutant IDH2 isoforms. Cells (1 × 106) were plated, lysed the next day and subsequently assayed for their ability to generate NADPH. (c) Caspase 3 activation was determined with DEVDase activity assay in stable U251 cells expressing wild-type and mutant IDH2 isoforms after 24 h ETO (50 μg/ml) exposure. Results are expressed relative to wild-type IDH2-expressing cells. (d) Caspase 3 activation was determined with DEVDase activity assay after 24 h ETO (50 μg/ml) exposure in U251 cells treated with D-2HG (3 mM) for 6 days. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH2, wild-type IDH2-expressing cells; R140, IDH1R140-expressing cells transfected; R172, IDH1R172-expressing cells transfected. *P<0.05, **P<0.01 and ***P<0.001
Mentions: To determine whether mutant IDH2 triggered similar effects on cell death, U251 cells were transfected with vector, IDH2, IDH2R140 and IDH2R172. Western blot analysis showed that IDH2 proteins were expressed in the mitochondria and not in the cytosol (Figure 3a). Similarly to IDH1R132, mutant IDH2-expressing cells exhibited decreased NADPH consumption corresponding to its neomorphic activity (Figure 3b). Interestingly, caspase-3 activation following ETO treatment was also significantly reduced (Figure 3c). Altogether, these results showed that all mutants IDH, independently of their respective subcellular localization, reduced ETO-induced cell death.

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