Increased Ammonium Toxicity in Response to Exogenous Glutamine in Metastatic Breast Cancer Cells

Abstract

Several cancers, including breast cancers, show dependence on glutamine metabolism. The purpose of the present study was to determine the mechanistic basis and impact of differential glutamine metabolism in nonmetastatic and metastatic murine mammary cancer cells. Universally labeled ¹³C₅-glutamine metabolic tracing, qRT-PCR, measures of reductive-oxidative balance, and exogenous ammonium chloride treatment were used to assess glutamine reprogramming. Results show that 4 mM media concentration of glutamine, compared with 2 mM, reduced viability only in metastatic cells, and that this decrease in viability was accompanied by increased incorporation of glutamine-derived carbon into the tricarboxylic acid (TCA) cycle. While increased glutamine metabolism in metastatic cells occurred in tandem with a decrease in the reduced/oxidized glutathione ratio, treatment with the antioxidant molecule N-acetylcysteine did not rescue cell viability. However, the viability of metastatic cells was more sensitive to ammonium chloride treatment compared with nonmetastatic cells, suggesting a role of metabolic reprogramming in averting nitrogen cytotoxicity in nonmetastatic cells. Overall, these results demonstrate the ability of nonmetastatic cancer cells to reprogram glutamine metabolism and that this ability may be lost in metastatic cells.

Keywords: ammonium toxicity; breast cancer; glutamine metabolism; metabolic reprogramming; metastasis.

Figure 1

Effect of glutamine concentration on glutamine metabolism. (A) Viability of M-Wnt and metM-Wnt^lung cells maintained in 2 mM or 4 mM glutamine was assessed by MTT; (B,C) mRNA level of genes involved in glutamine metabolism in M-Wnt and metM-Wnt^lung cells was determined; (D,E) labeled ¹³C₅-labeled glutamine was used to determine labeling of the downstream metabolites glutamate and α-ketoglutarate; (F) NADH/NAD⁺ ratios were measured. Overview of glutamine catabolism (G). Abbreviations: GLS—glutaminase; GLUL—glutamine synthetase; GLUD1—glutamate dehydrogenase; GOT2—glutamic oxaloacetic transaminase; GPT2—glutamic pyruvic transaminase; PSAT1—phosphoserine aminotransferase 1. The green box highlights transaminase reactions that are coupled with the conversion of glutamate to αKG; the grey box highlights ammonium-producing reactions. Grey circles indicate ¹³C labeled carbons in each metabolite if derived from exogenous universally labeled ¹³C glutamine. Results are expressed as means + SEM. Asterisk (*) indicates p < 0.05 relative to 2 mM glutamine.

Figure 2

Effect of glutamine concentration on oxidative stress markers. (A) GSH/GSSG and (B) NADPH/NADP⁺ ratios were measured in M-Wnt and metM-Wnt^lung cells; (C,D) ROS levels were assessed in M-Wnt and metM-Wnt^lung cells chronically grown in 2 mM or 4 mM glutamine, and in cells grown in 4 mM glutamine for indicated times; (E,F) the effects of N-acetylcysteine (NAC) on ROS and viability were assessed in cells grown in 4 mM glutamine. Results are expressed as means + SEM. Asterisk (*) indicates p < 0.05 relative to 2 mM glutamine (in A,D) or relative to 0 mM NAC (E,F).

Figure 3

Effect of dimethyl α-ketoglutarate and ammonium chloride on cell viability. (A) The effect of exogenous addition of a membrane-permeable form of α-ketoglutarate (dimethyl α-ketoglutarate, DMαKG) or (B) ammonium chloride (NH₄Cl) on viability was assessed in cells constitutively grown in 2 mM glutamine. Results are expressed as means + SEM. Asterisk (*) indicates p < 0.05 relative to 0 mM ammonium chloride (B).

Figure 4

Effect of glutamine concentration on ammonium detoxification genes. Relative mRNA level of carbamoyl-phosphate synthetase 2 (Cad) and asparagine synthetase (Asns) was determined by qRT-PCR in (A) M-Wnt and (B) metM-Wnt^lung cells grown in 2 or 4 mM glutamine.

Figure 5

Effect of ammonium chloride treatment on mRNA abundance. Relative mRNA level was determined by qRT-PCR in (A) M-Wnt and (B) metM-Wnt^lung cells grown in 2 mM glutamine with 1 mM ammonium chloride (NH₄Cl) or sodium chloride (Ctrl) for 48 h. Results are expressed as means + SEM. Asterisk (*) p < 0.05 relative to Ctrl.