Metabolic Analysis of Three-Dimensional Cultured Gastrointestinal Cancer Cells Suggests that <sc>l</sc>-Arginine Inhibits Tumor Growth by Affecting the Urea Cycle.

INTRODUCTION: There is evidence for the anticancer effects of l-arginine (arginine); however, the direct effects on cancer cells and mechanism of action are unclear. METHODS: Various upper gastrointestinal cancer cells (OE19, OE33, MKN1, MKN45, MKN74, and AGS) were divided into arginine-treated and -untreated groups and cultured using two-dimensional and three-dimensional culture systems. Proliferation was evaluated using the MTT assay to identify arginine-sensitive (OE33) and arginine-insensitive (OE19) strains. Furthermore, the effects of arginine were evaluated using a mitochondrial stress test, cell cycle assay, comprehensive metabolic analysis, and tracer study using (13C6) l-arginine. RESULTS: In OE33 (but not in OE19), the maximal respiratory capacity of mitochondria was lower in the treated group than in the control group. In OE33, S phase cells (determined using BrdU) were significantly reduced. In a comprehensive metabolic analysis of OE33, citrulline/ornithine levels were significantly lower in arginine-treated than in untreated cells. Using OE33, carbamoyl aspartic acid (CAA) levels were significantly lower in arginine-treated than in untreated cells. A tracer study suggested that arginine promotes the urea cycle. CONCLUSION: Arginine affected urea cycle metabolism, thereby decreasing CAA, which is required for pyrimidine nucleotide synthesis. These findings provide insight into the mechanism underlying the anticancer effects of arginine.