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Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine

View Article: PubMed Central - PubMed

ABSTRACT

Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by 13C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis.

No MeSH data available.


GLN starvation reduces PDH activity and increases PDK1 in the GLN addicted cells.(A) Western blot analysis of phospho-PDH levels in GLN addicted (U2OS, DU145) cells +/− GLN starved for 20 hours. Total PDH and Grb2 were used as loading controls. Blots were cropped for clarity. (B) PDH activity assay in U2OS and MCF7 cells +/− 20H GLN starvation. (C) Relative mRNA levels of four PDK isoforms in GLN addicted U2OS and DU145 cells and (D) in GLN independent MCF7 and PC3 cells +/− 20 H GLN starvation. (E) Effect of 2 mM dichloroacetate (DCA) on PDH activity in U2OS cells +/− 20 H GLN starvation. (F) Western blot analysis of cleaved-PARP, phospho-H2AX as cell death and oxidative stress markers, and phopsho-PDH in U2OS cells treated with indicated concentrations of DCA +/− 20 hour GLN starvation. Grb2 was used as loading control. Blots were cropped for clarity. Full length blots are given in Fig. S7. Data are average ± SD of 3 independent cultures. *P < 0.05, **P < 0.005, ***P < 0.001.
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f6: GLN starvation reduces PDH activity and increases PDK1 in the GLN addicted cells.(A) Western blot analysis of phospho-PDH levels in GLN addicted (U2OS, DU145) cells +/− GLN starved for 20 hours. Total PDH and Grb2 were used as loading controls. Blots were cropped for clarity. (B) PDH activity assay in U2OS and MCF7 cells +/− 20H GLN starvation. (C) Relative mRNA levels of four PDK isoforms in GLN addicted U2OS and DU145 cells and (D) in GLN independent MCF7 and PC3 cells +/− 20 H GLN starvation. (E) Effect of 2 mM dichloroacetate (DCA) on PDH activity in U2OS cells +/− 20 H GLN starvation. (F) Western blot analysis of cleaved-PARP, phospho-H2AX as cell death and oxidative stress markers, and phopsho-PDH in U2OS cells treated with indicated concentrations of DCA +/− 20 hour GLN starvation. Grb2 was used as loading control. Blots were cropped for clarity. Full length blots are given in Fig. S7. Data are average ± SD of 3 independent cultures. *P < 0.05, **P < 0.005, ***P < 0.001.

Mentions: Entry of glucose-derived carbons into the TCA cycle is regulated by the mitochondrial pyruvate dehydrogenase (PDH) complex, which catalyzes conversion of pyruvate to acetyl coenzyme A. Phosphorylation of PDH by pyruvate dehydrogenase kinases at the E1 subunit renders the PDH complex inactive3940. Since GLN addicted cells are defective in utilizing glucose in the TCA cycle in the absence of GLN, we predicted that PDH activity may be reduced by GLN starvation in GLN addicted cells. Using a phospho-specific PDH E1 subunit antibody, we analyzed the phospho-PDH levels in GLN addicted and GLN independent cells in the presence and absence of GLN by Western Blots. As shown in Fig. 6A, phospho-PDH levels increase only in the GLN addicted U2OS and DU145 cells upon GLN starvation, but not in the GLN independent MCF7 and PC3 cells (Fig. S6). We also compared the PDH enzyme activity in U2OS and MCF7 cells with or without GLN starvation. Consistent with increased phospho-PDH levels, PDH enzyme activity was significantly reduced (~50%) in GLN addicted U2OS cells compared to GLN independent MCF7 cells upon GLN starvation (Fig. 6B). Of note, U2OS cells exhibited ~2-fold lower PDH activity than MCF7 cells, which may explain why incorporation of glucose carbons into TCA cycle intermediates is lower in the U2OS cells as compared to MCF7 cells under GLN replete conditions. Since phosphorylation of PDH is regulated by PDK, we compared the expression levels of PDK isoforms in GLN addicted cells and GLN independent cells. PDK has four known isoforms with varying expression levels depending on the tissue and context41. Of the four isoforms, PDK1 expression was consistently increased in GLN addicted U2OS and DU145 cells upon GLN starvation, whereas in GLN independent MCF7 and PC3 cells it was unchanged or actually reduced (Fig. 6C,D). We then used dicholoroacetate (DCA), a well characterized PDK inhibitor42, in GLN addicted U2OS cells to determine whether inhibition of PDK increases PDH activity and rescues GLN starvation-induced oxidative stress and cell death. DCA treatment increased PDH activity (Fig. 6E) and partially reduced ROS levels and cell death based on phospho-H2AX and cleaved-PARP levels (Fig. 6F). Together these results suggest that inhibition of PDH activity by GLN starvation further reduces the entry of glucose-derived pyruvate carbons into the TCA cycle.


Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine
GLN starvation reduces PDH activity and increases PDK1 in the GLN addicted cells.(A) Western blot analysis of phospho-PDH levels in GLN addicted (U2OS, DU145) cells +/− GLN starved for 20 hours. Total PDH and Grb2 were used as loading controls. Blots were cropped for clarity. (B) PDH activity assay in U2OS and MCF7 cells +/− 20H GLN starvation. (C) Relative mRNA levels of four PDK isoforms in GLN addicted U2OS and DU145 cells and (D) in GLN independent MCF7 and PC3 cells +/− 20 H GLN starvation. (E) Effect of 2 mM dichloroacetate (DCA) on PDH activity in U2OS cells +/− 20 H GLN starvation. (F) Western blot analysis of cleaved-PARP, phospho-H2AX as cell death and oxidative stress markers, and phopsho-PDH in U2OS cells treated with indicated concentrations of DCA +/− 20 hour GLN starvation. Grb2 was used as loading control. Blots were cropped for clarity. Full length blots are given in Fig. S7. Data are average ± SD of 3 independent cultures. *P < 0.05, **P < 0.005, ***P < 0.001.
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f6: GLN starvation reduces PDH activity and increases PDK1 in the GLN addicted cells.(A) Western blot analysis of phospho-PDH levels in GLN addicted (U2OS, DU145) cells +/− GLN starved for 20 hours. Total PDH and Grb2 were used as loading controls. Blots were cropped for clarity. (B) PDH activity assay in U2OS and MCF7 cells +/− 20H GLN starvation. (C) Relative mRNA levels of four PDK isoforms in GLN addicted U2OS and DU145 cells and (D) in GLN independent MCF7 and PC3 cells +/− 20 H GLN starvation. (E) Effect of 2 mM dichloroacetate (DCA) on PDH activity in U2OS cells +/− 20 H GLN starvation. (F) Western blot analysis of cleaved-PARP, phospho-H2AX as cell death and oxidative stress markers, and phopsho-PDH in U2OS cells treated with indicated concentrations of DCA +/− 20 hour GLN starvation. Grb2 was used as loading control. Blots were cropped for clarity. Full length blots are given in Fig. S7. Data are average ± SD of 3 independent cultures. *P < 0.05, **P < 0.005, ***P < 0.001.
Mentions: Entry of glucose-derived carbons into the TCA cycle is regulated by the mitochondrial pyruvate dehydrogenase (PDH) complex, which catalyzes conversion of pyruvate to acetyl coenzyme A. Phosphorylation of PDH by pyruvate dehydrogenase kinases at the E1 subunit renders the PDH complex inactive3940. Since GLN addicted cells are defective in utilizing glucose in the TCA cycle in the absence of GLN, we predicted that PDH activity may be reduced by GLN starvation in GLN addicted cells. Using a phospho-specific PDH E1 subunit antibody, we analyzed the phospho-PDH levels in GLN addicted and GLN independent cells in the presence and absence of GLN by Western Blots. As shown in Fig. 6A, phospho-PDH levels increase only in the GLN addicted U2OS and DU145 cells upon GLN starvation, but not in the GLN independent MCF7 and PC3 cells (Fig. S6). We also compared the PDH enzyme activity in U2OS and MCF7 cells with or without GLN starvation. Consistent with increased phospho-PDH levels, PDH enzyme activity was significantly reduced (~50%) in GLN addicted U2OS cells compared to GLN independent MCF7 cells upon GLN starvation (Fig. 6B). Of note, U2OS cells exhibited ~2-fold lower PDH activity than MCF7 cells, which may explain why incorporation of glucose carbons into TCA cycle intermediates is lower in the U2OS cells as compared to MCF7 cells under GLN replete conditions. Since phosphorylation of PDH is regulated by PDK, we compared the expression levels of PDK isoforms in GLN addicted cells and GLN independent cells. PDK has four known isoforms with varying expression levels depending on the tissue and context41. Of the four isoforms, PDK1 expression was consistently increased in GLN addicted U2OS and DU145 cells upon GLN starvation, whereas in GLN independent MCF7 and PC3 cells it was unchanged or actually reduced (Fig. 6C,D). We then used dicholoroacetate (DCA), a well characterized PDK inhibitor42, in GLN addicted U2OS cells to determine whether inhibition of PDK increases PDH activity and rescues GLN starvation-induced oxidative stress and cell death. DCA treatment increased PDH activity (Fig. 6E) and partially reduced ROS levels and cell death based on phospho-H2AX and cleaved-PARP levels (Fig. 6F). Together these results suggest that inhibition of PDH activity by GLN starvation further reduces the entry of glucose-derived pyruvate carbons into the TCA cycle.

View Article: PubMed Central - PubMed

ABSTRACT

Cancer cells have altered metabolism compared to normal cells, including dependence on glutamine (GLN) for survival, known as GLN addiction. However, some cancer cell lines do not require GLN for survival and the basis for this discrepancy is not well understood. GLN is a precursor for antioxidants such as glutathione (GSH) and NADPH, and GLN deprivation is therefore predicted to deplete antioxidants and increase reactive oxygen species (ROS). Using diverse human cancer cell lines we show that this occurs only in cells that rely on GLN for survival. Thus, the preference for GLN as a dominant antioxidant source defines GLN addiction. We show that despite increased glucose uptake, GLN addicted cells do not metabolize glucose via the TCA cycle when GLN is depleted, as revealed by 13C-glucose labeling. In contrast, GLN independent cells can compensate by diverting glucose-derived pyruvate into the TCA cycle. GLN addicted cells exhibit reduced PDH activity, increased PDK1 expression, and PDK inhibition partially rescues GLN starvation-induced ROS and cell death. Finally, we show that combining GLN starvation with pro-oxidants selectively kills GLN addicted cells. These data highlight a major role for GLN in maintaining redox balance in cancer cells that lack glucose-dependent anaplerosis.

No MeSH data available.