Supplementary Material for: Metabolic analysis of three-dimensional cultured gastrointestinal cancer cells suggests that L-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, 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.