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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 severely reduces antioxidant pools in GLN addicted cells.GSH (A), NADPH (B), and GSSG (C) levels were measured in GLN addicted (DU145, U2OS) and GLN independent (MCF7, PC3) cells after 20 hours of GLN starvation. Data are the average ± SD of three independent cultures. *P < 0.05, ***P < 0.001.
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f2: GLN starvation severely reduces antioxidant pools in GLN addicted cells.GSH (A), NADPH (B), and GSSG (C) levels were measured in GLN addicted (DU145, U2OS) and GLN independent (MCF7, PC3) cells after 20 hours of GLN starvation. Data are the average ± SD of three independent cultures. *P < 0.05, ***P < 0.001.

Mentions: GLN is a precursor for production of GSH and NADPH, which are critical for managing ROS levels. Because GLN withdrawal increased ROS only in the GLN addicted cells, we predicted that depletion of GSH and NADPH pools should be more prominent in GLN addicted cells after GLN withdrawal. Indeed, levels of both GSH and NADPH were reduced by more than 50% in GLN addicted DU145 and U2OS cell lines after 24 hours of GLN starvation (Figs 2A and S3A), whereas only modest or non-significant changes were observed in the GLN independent cell lines, MCF7 and PC3 (Fig. 2B). Notably, addition of glutamate (GLU) significantly increased both GSH and NADPH levels in DU145 and U2OS cells (Fig. S3B,C). We also measured oxidized glutathione (GSSG) levels in the same cell lysates that were used in the GSH measurements, and found that GLN starvation significantly increases GSSG levels only in GLN addicted cells (Fig. 2C), indicating that GLN independent cells maintain sufficient levels of GSH and NADPH even after GLN starvation. Collectively, these findings suggest that GLN addicted cells rely predominantly on GLN for GSH and NADPH generation, whereas GLN independent cells likely utilize other precursors in addition to GLN.


Glucose-dependent anaplerosis in cancer cells is required for cellular redox balance in the absence of glutamine
GLN starvation severely reduces antioxidant pools in GLN addicted cells.GSH (A), NADPH (B), and GSSG (C) levels were measured in GLN addicted (DU145, U2OS) and GLN independent (MCF7, PC3) cells after 20 hours of GLN starvation. Data are the average ± SD of three independent cultures. *P < 0.05, ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC5015067&req=5

f2: GLN starvation severely reduces antioxidant pools in GLN addicted cells.GSH (A), NADPH (B), and GSSG (C) levels were measured in GLN addicted (DU145, U2OS) and GLN independent (MCF7, PC3) cells after 20 hours of GLN starvation. Data are the average ± SD of three independent cultures. *P < 0.05, ***P < 0.001.
Mentions: GLN is a precursor for production of GSH and NADPH, which are critical for managing ROS levels. Because GLN withdrawal increased ROS only in the GLN addicted cells, we predicted that depletion of GSH and NADPH pools should be more prominent in GLN addicted cells after GLN withdrawal. Indeed, levels of both GSH and NADPH were reduced by more than 50% in GLN addicted DU145 and U2OS cell lines after 24 hours of GLN starvation (Figs 2A and S3A), whereas only modest or non-significant changes were observed in the GLN independent cell lines, MCF7 and PC3 (Fig. 2B). Notably, addition of glutamate (GLU) significantly increased both GSH and NADPH levels in DU145 and U2OS cells (Fig. S3B,C). We also measured oxidized glutathione (GSSG) levels in the same cell lysates that were used in the GSH measurements, and found that GLN starvation significantly increases GSSG levels only in GLN addicted cells (Fig. 2C), indicating that GLN independent cells maintain sufficient levels of GSH and NADPH even after GLN starvation. Collectively, these findings suggest that GLN addicted cells rely predominantly on GLN for GSH and NADPH generation, whereas GLN independent cells likely utilize other precursors in addition to GLN.

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.