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Inhibition of Mitochondrial Complex II by the Anticancer Agent Lonidamine.

Guo L, Shestov AA, Worth AJ, Nath K, Nelson DS, Leeper DB, Glickson JD, Blair IA - J. Biol. Chem. (2015)

Bottom Line: However, the effect of LND on central energy metabolism has never been fully characterized.The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation.Using stable isotope tracers in combination with isotopologue analysis, we showed that LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate.

View Article: PubMed Central - PubMed

Affiliation: From the Penn Superfund Research and Training Program Center, Center of Excellence in Environmental Toxicology, and Department of Systems Pharmacology and Translational Therapeutics and.

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LND inhibits complex II activity in isolated mitochondria.A and B, isolated mouse liver mitochondria were incubated with DMSO (−) or the indicated drugs and 5 mm [13C4]succinate at 37 °C for 30 min. The levels of [13C4]fumarate (A) and [13C4]malate (B) generated in each treatment were normalized with respect to the relevant metabolites in the vehicle-treated group. The data presented are means of three samples. Error bars represent S.D. C and D, isolated mouse liver mitochondria were incubated with 5 mm succinate, 5 μm rotenone, and LND at the indicated concentrations. C, oxygen consumption was measured by a Clark electrode. Arrowheads indicate the addition of mitochondria, ADP, and FCCP. D, quantification of oxygen consumption rate in (C) expressed as nmol of oxygen/min/μg of mitochondrial protein.
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Figure 2: LND inhibits complex II activity in isolated mitochondria.A and B, isolated mouse liver mitochondria were incubated with DMSO (−) or the indicated drugs and 5 mm [13C4]succinate at 37 °C for 30 min. The levels of [13C4]fumarate (A) and [13C4]malate (B) generated in each treatment were normalized with respect to the relevant metabolites in the vehicle-treated group. The data presented are means of three samples. Error bars represent S.D. C and D, isolated mouse liver mitochondria were incubated with 5 mm succinate, 5 μm rotenone, and LND at the indicated concentrations. C, oxygen consumption was measured by a Clark electrode. Arrowheads indicate the addition of mitochondria, ADP, and FCCP. D, quantification of oxygen consumption rate in (C) expressed as nmol of oxygen/min/μg of mitochondrial protein.

Mentions: To rule out the possibility that the inhibition of the succinate dehydrogenase activity of complex II by LND was an indirect effect resulting from metabolic alterations in the cytosol, we examined the conversion of succinate to fumarate using isolated mitochondria. Importantly, the utilization of isolated mitochondria reduces interference of cytoplasmic metabolites and enzymes. To interrogate the specific action of complex II, isolated mitochondria were incubated with 5 mm [13C4]succinate. The generation of [13C4]fumarate, [13C4]malate, and [13C4]citrate was quantified to assess succinate metabolism. Within 30 min of incubation, 106 nmol of [13C4]fumarate and 232 nmol of [13C4]malate were generated from 60 μg of mitochondria with vehicle treatment. No heavy labeled citrate was detected, most likely due to the lack of external acetyl-CoA supply. The addition of LND inhibited the production of [13C4]fumarate and [13C4]malate (Fig. 2, A and B). 150 μm LND inhibited ∼40% of fumarate and 50% of malate production. The extent of inhibition caused by LND was similar to that of 3-NPA at various concentrations, whereas TTFA was a more potent inhibitor of complex II (Fig. 2, A and B). Overall, our observations suggest that the inhibition of complex II activity by LND is independent of lactate accumulation or other metabolic changes in the cytosol.


Inhibition of Mitochondrial Complex II by the Anticancer Agent Lonidamine.

Guo L, Shestov AA, Worth AJ, Nath K, Nelson DS, Leeper DB, Glickson JD, Blair IA - J. Biol. Chem. (2015)

LND inhibits complex II activity in isolated mitochondria.A and B, isolated mouse liver mitochondria were incubated with DMSO (−) or the indicated drugs and 5 mm [13C4]succinate at 37 °C for 30 min. The levels of [13C4]fumarate (A) and [13C4]malate (B) generated in each treatment were normalized with respect to the relevant metabolites in the vehicle-treated group. The data presented are means of three samples. Error bars represent S.D. C and D, isolated mouse liver mitochondria were incubated with 5 mm succinate, 5 μm rotenone, and LND at the indicated concentrations. C, oxygen consumption was measured by a Clark electrode. Arrowheads indicate the addition of mitochondria, ADP, and FCCP. D, quantification of oxygen consumption rate in (C) expressed as nmol of oxygen/min/μg of mitochondrial protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4697178&req=5

Figure 2: LND inhibits complex II activity in isolated mitochondria.A and B, isolated mouse liver mitochondria were incubated with DMSO (−) or the indicated drugs and 5 mm [13C4]succinate at 37 °C for 30 min. The levels of [13C4]fumarate (A) and [13C4]malate (B) generated in each treatment were normalized with respect to the relevant metabolites in the vehicle-treated group. The data presented are means of three samples. Error bars represent S.D. C and D, isolated mouse liver mitochondria were incubated with 5 mm succinate, 5 μm rotenone, and LND at the indicated concentrations. C, oxygen consumption was measured by a Clark electrode. Arrowheads indicate the addition of mitochondria, ADP, and FCCP. D, quantification of oxygen consumption rate in (C) expressed as nmol of oxygen/min/μg of mitochondrial protein.
Mentions: To rule out the possibility that the inhibition of the succinate dehydrogenase activity of complex II by LND was an indirect effect resulting from metabolic alterations in the cytosol, we examined the conversion of succinate to fumarate using isolated mitochondria. Importantly, the utilization of isolated mitochondria reduces interference of cytoplasmic metabolites and enzymes. To interrogate the specific action of complex II, isolated mitochondria were incubated with 5 mm [13C4]succinate. The generation of [13C4]fumarate, [13C4]malate, and [13C4]citrate was quantified to assess succinate metabolism. Within 30 min of incubation, 106 nmol of [13C4]fumarate and 232 nmol of [13C4]malate were generated from 60 μg of mitochondria with vehicle treatment. No heavy labeled citrate was detected, most likely due to the lack of external acetyl-CoA supply. The addition of LND inhibited the production of [13C4]fumarate and [13C4]malate (Fig. 2, A and B). 150 μm LND inhibited ∼40% of fumarate and 50% of malate production. The extent of inhibition caused by LND was similar to that of 3-NPA at various concentrations, whereas TTFA was a more potent inhibitor of complex II (Fig. 2, A and B). Overall, our observations suggest that the inhibition of complex II activity by LND is independent of lactate accumulation or other metabolic changes in the cytosol.

Bottom Line: However, the effect of LND on central energy metabolism has never been fully characterized.The ability of LND to promote cell death was potentiated by its suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation.Using stable isotope tracers in combination with isotopologue analysis, we showed that LND increased glutaminolysis but decreased reductive carboxylation of glutamine-derived α-ketoglutarate.

View Article: PubMed Central - PubMed

Affiliation: From the Penn Superfund Research and Training Program Center, Center of Excellence in Environmental Toxicology, and Department of Systems Pharmacology and Translational Therapeutics and.

Show MeSH
Related in: MedlinePlus