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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) Caspase 3 activation was determined with DEVDase activity assay in stable transfected U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms after induction of apoptosis. Cells were plated at 5 × 105 cells and treated the next day with different inducers of cell apoptosis. Cellular extracts were prepared from untreated cells (CTR), 5 h after treatment with TRAIL (50 ng/ml), 24 h after ETO (50 μg/ml), FASL (60ng/ml) or cisplatin (CIS) (15 μg/ml) and 72 h after γ-irradiation (IRR; 5 Gy). (b) The number of dead cells 24 h after ETO (50 μg/ml) treatment was determined by FACS. Cells were incubated 5 min with propidium iodide (1 μg/ml) and analyzed by FACS. (c) The mitochondrial membrane potential was determined by FACS after ETO (50 μg/ml) treatment at different time points. Cells were incubated 15 min with JC-1 probe and analyzed by FACS. (d and e) Caspase 3 activation after 24 h ETO (50 μg/ml) exposure was determined with DEVDase activity assay, respectively, in wild-type and mutant IDH1-overexpressing LN18 and T98 cells. Results are expressed relative to wild-type IDH1-expressing cells. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH1, wild-type IDH1-expressing cells; R132, IDH1R132-expressing cells transfected. *P<0.05, **P<0.01 and ***P<0.001
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fig2: (a) Caspase 3 activation was determined with DEVDase activity assay in stable transfected U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms after induction of apoptosis. Cells were plated at 5 × 105 cells and treated the next day with different inducers of cell apoptosis. Cellular extracts were prepared from untreated cells (CTR), 5 h after treatment with TRAIL (50 ng/ml), 24 h after ETO (50 μg/ml), FASL (60ng/ml) or cisplatin (CIS) (15 μg/ml) and 72 h after γ-irradiation (IRR; 5 Gy). (b) The number of dead cells 24 h after ETO (50 μg/ml) treatment was determined by FACS. Cells were incubated 5 min with propidium iodide (1 μg/ml) and analyzed by FACS. (c) The mitochondrial membrane potential was determined by FACS after ETO (50 μg/ml) treatment at different time points. Cells were incubated 15 min with JC-1 probe and analyzed by FACS. (d and e) Caspase 3 activation after 24 h ETO (50 μg/ml) exposure was determined with DEVDase activity assay, respectively, in wild-type and mutant IDH1-overexpressing LN18 and T98 cells. Results are expressed relative to wild-type IDH1-expressing cells. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH1, wild-type IDH1-expressing cells; R132, IDH1R132-expressing cells transfected. *P<0.05, **P<0.01 and ***P<0.001

Mentions: Cell death was measured at different time points after irradiation (5 Gy), ETO (50 μg/ml), TRAIL (50 ng/ml), FasL (60 ng/ml) or cisplatin (15 μg/ml) treatment in U251 cells. All treatments were associated with a significant death and activation of caspase-3 (Table 1). However, the optimal time point of cell death induction varied from 6 h with TRAIL to 24 h with ETO, Cisplatin, and FASL treatments, and to 72 h with irradiation. Next, sensitivity to cell death was analyzed in IDH1- and IDH1R132-overexpressing cells. For most treatments, overexpression of IDH1R132 did not affect caspase-3 activity (Figure 2a). However, although addition of ETO caused a high caspase-3 activation in control and wild-type IDH1-overexpressing cells, activation of caspase-3 was significantly reduced in IDH1R132-overexpressing cells. These results were confirmed by FACs analysis, which showed that the percentage of propidium iodide-stained cells after ETO exposure was lower in IDH1R132 cells compared with the control and IDH1 cells (Figure 2b). During apoptosis, the integrity of the mitochondrial outer membrane is compromised, a process called mitochondrial outer membrane permeabilization (MOMP). In order to determine whether IDH1 or IDH1R132 expression was affecting the MOMP, we measured the mitochondrial membrane potential ΔΨm in our cells using JC-1 staining. ΔΨm was reduced in a time-dependent manner after ETO exposure as indicated by the decrease of the red/green ratio (Figure 2c). Of note, the median of healthy cells red fluorescence was significantly higher in IDH1R132 cells compared with IDH1 cells (845±75 versus 655±56; n=6; P<0.05), suggesting a mitochondrial hyperpolarization of IDH1R132 cells. Sensitivity to ETO was then tested in other glioma cell lines overexpressing either IDH1 or IDH1R132, LN18 and T98 (Figures 2d and e). In both cells lines, IDH1R132 overexpression was associated with reduced ETO-induced cell apoptosis.


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) Caspase 3 activation was determined with DEVDase activity assay in stable transfected U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms after induction of apoptosis. Cells were plated at 5 × 105 cells and treated the next day with different inducers of cell apoptosis. Cellular extracts were prepared from untreated cells (CTR), 5 h after treatment with TRAIL (50 ng/ml), 24 h after ETO (50 μg/ml), FASL (60ng/ml) or cisplatin (CIS) (15 μg/ml) and 72 h after γ-irradiation (IRR; 5 Gy). (b) The number of dead cells 24 h after ETO (50 μg/ml) treatment was determined by FACS. Cells were incubated 5 min with propidium iodide (1 μg/ml) and analyzed by FACS. (c) The mitochondrial membrane potential was determined by FACS after ETO (50 μg/ml) treatment at different time points. Cells were incubated 15 min with JC-1 probe and analyzed by FACS. (d and e) Caspase 3 activation after 24 h ETO (50 μg/ml) exposure was determined with DEVDase activity assay, respectively, in wild-type and mutant IDH1-overexpressing LN18 and T98 cells. Results are expressed relative to wild-type IDH1-expressing cells. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH1, wild-type IDH1-expressing cells; R132, IDH1R132-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
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fig2: (a) Caspase 3 activation was determined with DEVDase activity assay in stable transfected U251 cells expressing empty vector, wild-type and mutant IDH1 isoforms after induction of apoptosis. Cells were plated at 5 × 105 cells and treated the next day with different inducers of cell apoptosis. Cellular extracts were prepared from untreated cells (CTR), 5 h after treatment with TRAIL (50 ng/ml), 24 h after ETO (50 μg/ml), FASL (60ng/ml) or cisplatin (CIS) (15 μg/ml) and 72 h after γ-irradiation (IRR; 5 Gy). (b) The number of dead cells 24 h after ETO (50 μg/ml) treatment was determined by FACS. Cells were incubated 5 min with propidium iodide (1 μg/ml) and analyzed by FACS. (c) The mitochondrial membrane potential was determined by FACS after ETO (50 μg/ml) treatment at different time points. Cells were incubated 15 min with JC-1 probe and analyzed by FACS. (d and e) Caspase 3 activation after 24 h ETO (50 μg/ml) exposure was determined with DEVDase activity assay, respectively, in wild-type and mutant IDH1-overexpressing LN18 and T98 cells. Results are expressed relative to wild-type IDH1-expressing cells. Results are expressed as the mean±S.E.M. of three experiments performed in triplicate. V, empty vector expressing cells; IDH1, wild-type IDH1-expressing cells; R132, IDH1R132-expressing cells transfected. *P<0.05, **P<0.01 and ***P<0.001
Mentions: Cell death was measured at different time points after irradiation (5 Gy), ETO (50 μg/ml), TRAIL (50 ng/ml), FasL (60 ng/ml) or cisplatin (15 μg/ml) treatment in U251 cells. All treatments were associated with a significant death and activation of caspase-3 (Table 1). However, the optimal time point of cell death induction varied from 6 h with TRAIL to 24 h with ETO, Cisplatin, and FASL treatments, and to 72 h with irradiation. Next, sensitivity to cell death was analyzed in IDH1- and IDH1R132-overexpressing cells. For most treatments, overexpression of IDH1R132 did not affect caspase-3 activity (Figure 2a). However, although addition of ETO caused a high caspase-3 activation in control and wild-type IDH1-overexpressing cells, activation of caspase-3 was significantly reduced in IDH1R132-overexpressing cells. These results were confirmed by FACs analysis, which showed that the percentage of propidium iodide-stained cells after ETO exposure was lower in IDH1R132 cells compared with the control and IDH1 cells (Figure 2b). During apoptosis, the integrity of the mitochondrial outer membrane is compromised, a process called mitochondrial outer membrane permeabilization (MOMP). In order to determine whether IDH1 or IDH1R132 expression was affecting the MOMP, we measured the mitochondrial membrane potential ΔΨm in our cells using JC-1 staining. ΔΨm was reduced in a time-dependent manner after ETO exposure as indicated by the decrease of the red/green ratio (Figure 2c). Of note, the median of healthy cells red fluorescence was significantly higher in IDH1R132 cells compared with IDH1 cells (845±75 versus 655±56; n=6; P<0.05), suggesting a mitochondrial hyperpolarization of IDH1R132 cells. Sensitivity to ETO was then tested in other glioma cell lines overexpressing either IDH1 or IDH1R132, LN18 and T98 (Figures 2d and e). In both cells lines, IDH1R132 overexpression was associated with reduced ETO-induced cell apoptosis.

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