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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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Effect of LND on GSH, NADPH, and pentose phosphate pathway.A, GSH levels of DB-1 cells treated with DMSO or the indicated drugs were quantified by LC-MS. B, a scheme showing the links among glycolysis, the PPP, NADPH production, and GSH redox cycling. G6PDH, glucose-6-phospate dehydrogenase; 6-PGDH, 6-phosphogluconate dehydrogenase; GL, gluconolactonase; GP, glutathione peroxidase; GR, glutathione reductase; GSSG, glutathione disulfide; LDH, lactate dehydrogenase; PFK-1, phosphofructokinase 1; PGI, phosphoglucose isomerase; HK, hexokinase. C and D, cells treated with DMSO, LND (150 μm), or TTFA (150 μm) for 4 h. NADPH and NADP+ were measured by LC-MS. Shown are the relative levels of NADPH (C) and relative ratios of NADPH/NADP+ (D). E, DB-1 cells were incubated with DMSO or LND (150 μm) for 1 h. Levels of 6-phoshogluconate (6-PG) and fructose 1,6-bisphosphate (Fru-1,6-BP) were measured by LC-MS. F and G, DB-1 cells treated with DMSO or LND (150 μm) were labeled with [13C3]glucose for the indicated times. Shown are the 13C labeling percentages of 6-phosphogluconate (F) and fructose 1,6-bisphosphate (G) plotted over time. p values between the control and LND-treated groups were calculated by two-way analysis of variance with repeated measures. For A and C–G, the means of three samples are shown. Error bars represent S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Figure 5: Effect of LND on GSH, NADPH, and pentose phosphate pathway.A, GSH levels of DB-1 cells treated with DMSO or the indicated drugs were quantified by LC-MS. B, a scheme showing the links among glycolysis, the PPP, NADPH production, and GSH redox cycling. G6PDH, glucose-6-phospate dehydrogenase; 6-PGDH, 6-phosphogluconate dehydrogenase; GL, gluconolactonase; GP, glutathione peroxidase; GR, glutathione reductase; GSSG, glutathione disulfide; LDH, lactate dehydrogenase; PFK-1, phosphofructokinase 1; PGI, phosphoglucose isomerase; HK, hexokinase. C and D, cells treated with DMSO, LND (150 μm), or TTFA (150 μm) for 4 h. NADPH and NADP+ were measured by LC-MS. Shown are the relative levels of NADPH (C) and relative ratios of NADPH/NADP+ (D). E, DB-1 cells were incubated with DMSO or LND (150 μm) for 1 h. Levels of 6-phoshogluconate (6-PG) and fructose 1,6-bisphosphate (Fru-1,6-BP) were measured by LC-MS. F and G, DB-1 cells treated with DMSO or LND (150 μm) were labeled with [13C3]glucose for the indicated times. Shown are the 13C labeling percentages of 6-phosphogluconate (F) and fructose 1,6-bisphosphate (G) plotted over time. p values between the control and LND-treated groups were calculated by two-way analysis of variance with repeated measures. For A and C–G, the means of three samples are shown. Error bars represent S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Mentions: To investigate why the LND-treated cells were more susceptible to complex II inhibition than TTFA-treated cells, levels of the cellular antioxidant GSH were compared. LND caused a 40% drop in GSH levels at a concentration of 150 μm and above (Fig. 5A). The extent of GSH reduction is similar to that caused by 100 μm diethyl maleate (DEM), a compound known to deplete GSH (Fig. 5A). In contrast, TTFA (50 or 200 μm) caused a modest reduction of 6% (Fig. 5A). Consistent with the reduced GSH levels, the levels of NADPH and the NADPH/NADP+ ratio both decreased after LND treatment but not after TTFA treatment (Fig. 5, C and D). The PPP is an important source of NADPH (5) required for the GSH reductase-mediated reduction of glutathione disulfide back to GSH (42). LND has been reported previously to be an inhibitor of hexokinase (Fig. 5B) (23, 43), which catalyzes the first step of glycolysis. In contrast, some studies have shown no effect on hexokinase based on the analysis of glucose 6-phosphate after LND treatment of breast cancer and glioma cell lines (25, 26, 43). However, these latter studies did not examine the effect of LND on specific PPP metabolites, which could also be down-regulated as a result of hexokinase inhibition (Fig. 5B). In support of this possibility, we found that the concentration of 6-phosphogluconate, an important PPP metabolite, was markedly decreased in LND-treated DB-1 cells (Fig. 5E). In addition, a time course for the incorporation of [13C6]glucose into PPP metabolites (determined by LC-MS) revealed that incorporation into the M + 6 isotopologue [13C6]6-phosphogluconate as well as the glycolysis metabolite [13C6]fructose 1,6-bisphosphate was significantly delayed (Fig. 5, F and G). These data, together with the reduced levels of 6-phosphogluconate, suggest that the flux into the PPP was greatly reduced by LND, most likely through inhibition of hexokinase. Thus, the reduced NADPH levels (Fig. 5C) and NADPH/NADP+ ratios (Fig. 5D) and decreased GSH levels (Fig. 5A) in LND-treated cells resulted, in part, from inhibition of the PPP (Fig. 5B).


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)

Effect of LND on GSH, NADPH, and pentose phosphate pathway.A, GSH levels of DB-1 cells treated with DMSO or the indicated drugs were quantified by LC-MS. B, a scheme showing the links among glycolysis, the PPP, NADPH production, and GSH redox cycling. G6PDH, glucose-6-phospate dehydrogenase; 6-PGDH, 6-phosphogluconate dehydrogenase; GL, gluconolactonase; GP, glutathione peroxidase; GR, glutathione reductase; GSSG, glutathione disulfide; LDH, lactate dehydrogenase; PFK-1, phosphofructokinase 1; PGI, phosphoglucose isomerase; HK, hexokinase. C and D, cells treated with DMSO, LND (150 μm), or TTFA (150 μm) for 4 h. NADPH and NADP+ were measured by LC-MS. Shown are the relative levels of NADPH (C) and relative ratios of NADPH/NADP+ (D). E, DB-1 cells were incubated with DMSO or LND (150 μm) for 1 h. Levels of 6-phoshogluconate (6-PG) and fructose 1,6-bisphosphate (Fru-1,6-BP) were measured by LC-MS. F and G, DB-1 cells treated with DMSO or LND (150 μm) were labeled with [13C3]glucose for the indicated times. Shown are the 13C labeling percentages of 6-phosphogluconate (F) and fructose 1,6-bisphosphate (G) plotted over time. p values between the control and LND-treated groups were calculated by two-way analysis of variance with repeated measures. For A and C–G, the means of three samples are shown. Error bars represent S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
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Figure 5: Effect of LND on GSH, NADPH, and pentose phosphate pathway.A, GSH levels of DB-1 cells treated with DMSO or the indicated drugs were quantified by LC-MS. B, a scheme showing the links among glycolysis, the PPP, NADPH production, and GSH redox cycling. G6PDH, glucose-6-phospate dehydrogenase; 6-PGDH, 6-phosphogluconate dehydrogenase; GL, gluconolactonase; GP, glutathione peroxidase; GR, glutathione reductase; GSSG, glutathione disulfide; LDH, lactate dehydrogenase; PFK-1, phosphofructokinase 1; PGI, phosphoglucose isomerase; HK, hexokinase. C and D, cells treated with DMSO, LND (150 μm), or TTFA (150 μm) for 4 h. NADPH and NADP+ were measured by LC-MS. Shown are the relative levels of NADPH (C) and relative ratios of NADPH/NADP+ (D). E, DB-1 cells were incubated with DMSO or LND (150 μm) for 1 h. Levels of 6-phoshogluconate (6-PG) and fructose 1,6-bisphosphate (Fru-1,6-BP) were measured by LC-MS. F and G, DB-1 cells treated with DMSO or LND (150 μm) were labeled with [13C3]glucose for the indicated times. Shown are the 13C labeling percentages of 6-phosphogluconate (F) and fructose 1,6-bisphosphate (G) plotted over time. p values between the control and LND-treated groups were calculated by two-way analysis of variance with repeated measures. For A and C–G, the means of three samples are shown. Error bars represent S.D. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
Mentions: To investigate why the LND-treated cells were more susceptible to complex II inhibition than TTFA-treated cells, levels of the cellular antioxidant GSH were compared. LND caused a 40% drop in GSH levels at a concentration of 150 μm and above (Fig. 5A). The extent of GSH reduction is similar to that caused by 100 μm diethyl maleate (DEM), a compound known to deplete GSH (Fig. 5A). In contrast, TTFA (50 or 200 μm) caused a modest reduction of 6% (Fig. 5A). Consistent with the reduced GSH levels, the levels of NADPH and the NADPH/NADP+ ratio both decreased after LND treatment but not after TTFA treatment (Fig. 5, C and D). The PPP is an important source of NADPH (5) required for the GSH reductase-mediated reduction of glutathione disulfide back to GSH (42). LND has been reported previously to be an inhibitor of hexokinase (Fig. 5B) (23, 43), which catalyzes the first step of glycolysis. In contrast, some studies have shown no effect on hexokinase based on the analysis of glucose 6-phosphate after LND treatment of breast cancer and glioma cell lines (25, 26, 43). However, these latter studies did not examine the effect of LND on specific PPP metabolites, which could also be down-regulated as a result of hexokinase inhibition (Fig. 5B). In support of this possibility, we found that the concentration of 6-phosphogluconate, an important PPP metabolite, was markedly decreased in LND-treated DB-1 cells (Fig. 5E). In addition, a time course for the incorporation of [13C6]glucose into PPP metabolites (determined by LC-MS) revealed that incorporation into the M + 6 isotopologue [13C6]6-phosphogluconate as well as the glycolysis metabolite [13C6]fructose 1,6-bisphosphate was significantly delayed (Fig. 5, F and G). These data, together with the reduced levels of 6-phosphogluconate, suggest that the flux into the PPP was greatly reduced by LND, most likely through inhibition of hexokinase. Thus, the reduced NADPH levels (Fig. 5C) and NADPH/NADP+ ratios (Fig. 5D) and decreased GSH levels (Fig. 5A) in LND-treated cells resulted, in part, from inhibition of the PPP (Fig. 5B).

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