Metabolic Fingerprinting of Murine L929 Fibroblasts as a Cell-Based Tumour Suppressor Model System for Methionine Restriction

Abstract

Since Otto Warburg reported in 1924 that cancer cells address their increased energy requirement through a massive intake of glucose, the cellular energy level has offered a therapeutic anticancer strategy. Methionine restriction (MetR) is one of the most effective approaches for inducing low-energy metabolism (LEM) due to the central position in metabolism of this amino acid. However, no simple in vitro system for the rapid analysis of MetR is currently available, and this study establishes the murine cell line L929 as such a model system. L929 cells react rapidly and efficiently to MetR, and the analysis of more than 150 different metabolites belonging to different classes (amino acids, urea and tricarboxylic acid cycle (TCA) cycles, carbohydrates, etc.) by liquid chromatography/mass spectrometry (LC/MS) defines a metabolic fingerprint and enables the identification of specific metabolites representing normal or MetR conditions. The system facilitates the rapid and efficient testing of potential cancer therapeutic metabolic targets. To date, MS studies of MetR have been performed using organisms and yeast, and the current LC/MS analysis of the intra- and extracellular metabolites in the murine cell line L929 over a period of 5 days thus provides new insights into the effects of MetR at the cellular metabolic level.

Low Energy Status under Methionine Restriction Is Essentially Independent of Proliferation or Cell Contact Inhibition

“…The overall results are shown in the sup plements (Tables S1 and S2) for all experimental series. In addition, the results were com pared with the results of a previous work in which the experiments were performed unde proliferative conditions [20]. 3.1.…”

“…In a previous work, we established the murine cell line L929 as a model system to analyse MetR [20]. Thus, we deliberately chose a murine cell line for several reasons.…”

“…The overall results are shown in the Supplements (Tables S1 and S2) for all experimental series. In addition, the results were compared with the results of a previous work in which the experiments were performed under proliferative conditions [20]. To determine how contact inhibition under full medium and under MetR affects me tabolism, we seeded an appropriate number of cells on cell culture dishes and incubated them for 48 h before stimulation until the cells formed a confluent cell layer.…”

“…For cell confluence, 3 × 10 6 cells were seeded, and after 48 h, cells were stimulated with complete or Met(−) media. In an earlier work, 1 × 10 6 cells/Petri dish were seeded under proliferative conditions for days 1, 2, and 3, and 5 × 10 5 cells were seeded for days 4 and 5 to prevent confluence during the test period [20]. The media used for stimulation were prepared from methionine-free RPMI medium.…”

“…by liquid chromatography/mass spectrometry (LC/MS) defines a metabolic fingerprint and enables the identification of specific metabolites representing normal or MetR conditions. In addition to the large fingerprint with numerous metabolites, the induction of LEM can potentially be analysed using a small footprint with selected metabolites that are specific/characteristic of MetR-induced LEM and are composed of the combination of acetoacetate, creatine, spermidine, GSSG, UDP-glucose, pantothenate, and ATP [20].…”