In vitro effect of extracellular AMP on MCF‐7 breast cancer cells: Inhibition of glycolysis and cell proliferation

Abstract

MCF-7 human breast cancer cells propagated in vitro were treated with adenosine derivatives added to the culture medium. The effects on cell proliferation, glycolysis, and glutaminolysis were investigated. Of all adenosine derivatives tested, AMP was the most efficient inhibitor of cell proliferation. In AMP-treated cells, DNA synthesis decreased, whereas RNA and protein syntheses rose normally with time. In terms of carbohydrate metabolism, lactate production from glucose was drastically reduced; therefore, most of lactate produced must have been derived from glutamine. Increases in the enzyme activities involved in glutamate degradation and in the malate-aspartate shuttle were observed. In contrast, actual glycolytic flux rates declined, whereas key glycolytic enzyme activities increased. Metabolites such as fructose 1,6-bisphosphate and pyruvate accumulated in AMP-arrested cells. Based on the lowered NAD level in the AMP-treated cells, lactate dehydrogenase, but not malate dehydrogenase, was impaired; thereby the whole of glycolysis was inhibited. In compensation, glutamine catabolism was increased. NAD concentrations fell drastically because of the known inhibition of P-ribose-PP synthesis through heightened intracellular AMP levels. A hypothetical metabolic scheme to explain these results and to show how extracellular AMP may influence carbohydrate metabolism and cell proliferation is presented.

Effect of Extracellular AMP on Cell Proliferation and Metabolism of Breast Cancer Cell Lines with High and Low Glycolytic Rates

“…The cells were removed from the plates with trypsin/EDTA from Biochrom, Berlin, Germany, and counted in a hemocytometer. The frozen supernatants were heated for 15 min at 80°C and were subsequently centrifuged at 8000 ϫ g for 10 min (49). Glucose, lactate, pyruvate, glutamine, and glutamate concentrations were determined according to Bergmeyer (60).…”

“…For the extraction of intracellular lactate, pyruvate, and NAD, cells were treated at 80°C in aqua bidest for 15 min as described previously (49). The concentrations of lactate, pyruvate, and NAD were measured enzymatically (60 (47).…”

“…The extractions and the measurements of enzyme activities were carried out as described in Ref. 49. Protein concentrations were measured by the Biuret method using a commercially available test kit from Boehringer Mannheim, Germany.…”

“…For MCF-7 cells this was basic DMEM supplemented with 5 mM glucose and for MDA-MB-453 the basic DMEM supplemented with 5 mM galactose and 0.5 mM glucose (from FCS). The concentration of AMP depends on the content of adenosine deaminase in the FCS charge (49). Adenosine deaminase degrades AMP to inosine monophosphate, which has no inhibitory effect on the proliferation rate of the two cell lines (49).…”

“…NADH levels drop so low that lactate dehydrogenase is no longer able to transfer the hydrogen from NADH to pyruvate. As a consequence, glycolysis is inhibited at the level of the NADH producing glyceraldehyde-3-phosphate dehydrogenase reaction (49). The metabolic behavior of MCF-7 cells is in complete contrast to differentiated tissues and cells where the increase of AMP under hypoxic conditions drastically activates 6-phosphofructo-1-kinase and the glycolytic flux rate (55)(56)(57)(58)(59).…”