Targeted Metabolomic Profiling of Free Amino Acids in Human Colon Tumor-116 and Normal Colon Cells

INTRODUCTION: Metabolic reprogramming is a defining feature of colorectal cancer, and amino acid metabolism constitutes a major functional axis supporting proliferation, stress tolerance, and treatment resistance. We aimed to compare the intracellular free amino acid profile of human colon tumor-116 (HCT-116) colon cancer cells with that of normal colon cells (CCD) using a targeted metabolomics workflow.
METHODS: Cells were cultured under standard conditions. Free amino acids were quantified by liquid chromatography-mass spectrometry/mass spectrometry using multiple reaction monitoring following derivatization with a commercial kit, based on a modified protocol previously described by Celik et al. Data preprocessing (median normalization, log10 transformation, and Pareto scaling) and statistical analyses were performed in MetaboAnalyst 6.0. Group comparisons used a two-sample t-test with Benjamini-Hochberg false discovery rate correction. Multivariate analyses (principal component analysis [PCA] and partial least squares-discriminant analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment/topology analyses were conducted.
RESULTS: PCA demonstrated clear metabolic separation between HCT-116 and CCD cells, with PC1 explaining 91.1% of total variance. Differential analysis indicated selective yet pronounced changes: taurine and phosphoethanolamine were increased, whereas citrulline, cystine, and thiaproline were decreased in HCT-116 cells. KEGG pathway analysis highlighted significant enrichment of valine/leucine/isoleucine biosynthesis, one-carbon pool by folate, arginine biosynthesis, and glycine/serine/threonine metabolism.
DISCUSSION AND CONCLUSION: HCT-116 cells exhibited pronounced reprogramming across redox-related (cystine/taurine), membrane phospholipid intermediate (phosphoethanolamine), and one-carbon/arginine-linked metabolic networks. This study supports hypothesis generation for testing targetable vulnerabilities, including the xCT/SLC7A11 axis and key one-carbon metabolic nodes.

Keywords: Amino acid metabolism, colorectal cancer, cystine, metabolomic, taurine.