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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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Dynamic labeling through oxidative glutamine metabolism in LND- and TTFA-treated cells. Dynamic labeling of DMSO-, LND- (150 μm), and TTFA (50 μm)-treated cells after switching to [13C5,15N2]glutamine. Data were collected from the same dynamic labeling experiment as described in Fig. 7. Labeling percentages of M + 4 succinate (A), fumarate (B), malate (C), and citrate (D) were plotted over time. The data presented are the means of three samples. Error bars represent S.D.
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Figure 8: Dynamic labeling through oxidative glutamine metabolism in LND- and TTFA-treated cells. Dynamic labeling of DMSO-, LND- (150 μm), and TTFA (50 μm)-treated cells after switching to [13C5,15N2]glutamine. Data were collected from the same dynamic labeling experiment as described in Fig. 7. Labeling percentages of M + 4 succinate (A), fumarate (B), malate (C), and citrate (D) were plotted over time. The data presented are the means of three samples. Error bars represent S.D.

Mentions: In addition to the effect on ROS generation and cell viability, we also further explored the metabolic changes triggered by LND and complex II inhibition. Glutamine is an important carbon source for TCA cycle anaplerosis in many cancer cells (Fig. 7A) (44). Therefore, we examined the effect of LND on glutamine metabolism and compared it with TTFA at a lower concentration so that they both induce a similar extent of succinate accumulation (Fig. 7B). Five carbon atoms from glutamine enter the TCA cycle via α-ketoglutarate followed by conversion to succinyl-CoA and succinate (Fig. 7A). Alternatively, α-ketoglutarate can be metabolized reductively to citrate to provide carbon atoms for lipid synthesis (Fig. 7A). We performed a dynamic labeling assay to analyze both oxidative and reductive pathways of glutamine metabolism. DB-1 cells were first treated with vehicle, LND, or TTFA and then cultured in medium containing [13C5,15N2]glutamine. Incorporation of [13C]carbons into TCA cycle metabolites at different time points was then determined. Over the 6 h of labeling, glutamine contributed to succinate, fumarate, malate, and citrate through oxidative metabolism as shown by their M + 4 isotopologues (Figs. 7 and 8). Most metabolites reached steady state labeling beyond 4 h in control cells (Fig. 8). Treatment of DB-1 cells with LND strikingly increased the labeling in succinate (Fig. 8A), fumarate (Fig. 8B), malate (Fig. 8C), and citrate (Fig. 8D) over the time course of the experiment as well as at steady state (Fig. 7, C–F). Similar effects were also observed in TTFA-treated cells (Figs. 7, C–F, and 8, A–D).


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)

Dynamic labeling through oxidative glutamine metabolism in LND- and TTFA-treated cells. Dynamic labeling of DMSO-, LND- (150 μm), and TTFA (50 μm)-treated cells after switching to [13C5,15N2]glutamine. Data were collected from the same dynamic labeling experiment as described in Fig. 7. Labeling percentages of M + 4 succinate (A), fumarate (B), malate (C), and citrate (D) were plotted over time. The data presented are the means of three samples. Error bars represent S.D.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Dynamic labeling through oxidative glutamine metabolism in LND- and TTFA-treated cells. Dynamic labeling of DMSO-, LND- (150 μm), and TTFA (50 μm)-treated cells after switching to [13C5,15N2]glutamine. Data were collected from the same dynamic labeling experiment as described in Fig. 7. Labeling percentages of M + 4 succinate (A), fumarate (B), malate (C), and citrate (D) were plotted over time. The data presented are the means of three samples. Error bars represent S.D.
Mentions: In addition to the effect on ROS generation and cell viability, we also further explored the metabolic changes triggered by LND and complex II inhibition. Glutamine is an important carbon source for TCA cycle anaplerosis in many cancer cells (Fig. 7A) (44). Therefore, we examined the effect of LND on glutamine metabolism and compared it with TTFA at a lower concentration so that they both induce a similar extent of succinate accumulation (Fig. 7B). Five carbon atoms from glutamine enter the TCA cycle via α-ketoglutarate followed by conversion to succinyl-CoA and succinate (Fig. 7A). Alternatively, α-ketoglutarate can be metabolized reductively to citrate to provide carbon atoms for lipid synthesis (Fig. 7A). We performed a dynamic labeling assay to analyze both oxidative and reductive pathways of glutamine metabolism. DB-1 cells were first treated with vehicle, LND, or TTFA and then cultured in medium containing [13C5,15N2]glutamine. Incorporation of [13C]carbons into TCA cycle metabolites at different time points was then determined. Over the 6 h of labeling, glutamine contributed to succinate, fumarate, malate, and citrate through oxidative metabolism as shown by their M + 4 isotopologues (Figs. 7 and 8). Most metabolites reached steady state labeling beyond 4 h in control cells (Fig. 8). Treatment of DB-1 cells with LND strikingly increased the labeling in succinate (Fig. 8A), fumarate (Fig. 8B), malate (Fig. 8C), and citrate (Fig. 8D) over the time course of the experiment as well as at steady state (Fig. 7, C–F). Similar effects were also observed in TTFA-treated cells (Figs. 7, C–F, and 8, A–D).

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