Assessment of Anticancer Drug Effects on Pancreatic Cancer Cells under Glucose-Depleted Conditions Using Intracellular and Extracellular Amino Acid Metabolomics

Abstract

Previously, we developed a method to evaluate states of cells treated with anticancer drugs via the comprehensive analysis of amino acids, termed amino acid metabolomics. In the present study, we evaluated the effects of the anticancer drugs, gemcitabine hydrochloride and pyrvinium pamoate, on the proliferation of a pancreatic cancer cell line (PANC-1) under hypoglycemic conditions using amino acid metabolomics. Intracellular and extracellular amino acid profiles of PANC-1 were determined by hydrophilic interaction chromatography-tandem mass spectrometry with simple pretreatment. Changes to the drugs' anticancer effects resulting from glucose starvation conditions were presented in score plots obtained from principal component analyses. In particular, the analysis of intracellular amino acids was found to be the superior approach because the results allowed a clearer assessment of the cell state. Further, orthogonal partial least squares discriminant analysis was performed to search for amino acid candidates that discriminate with anticancer drug-treated PANC-1 cells. We identified several amino acids that might be able to distinguish the drugtreated group from the control group. These results might provide a better understanding of the mechanisms underlying cell responses such as drug resistance or austerity. The present study is the first to evaluate the efficacy of anticancer drugs under glucose starvation based on the analysis of the variation of extracellular and intracellular amino acid profiles in vitro. Key words amino acid metabolomics; pancreatic cancer cell; glucose depletion; biomarker candidateMetabolomics is a comprehensive analysis of numerous small endogenous molecules contained in cells, biological fluids, and tissues. In addition to a complementary understanding of physiological functions, metabolomics is also a promising tool to explore potential biomarkers for diagnosis, prognosis, and therapeutic response.1) It has proven to be very useful not only in medical and life sciences, but also in clinical research, 2,3) drug discovery, 4) environmental toxicology, 5) and food science. 6,7) Recently, among the available focused metabolomics approaches, amino acid metabolomics analysis, which focuses on amino acids as key metabolites, such as aminoindex 8,9) or chiral amino acid metabolomics, 10-12) has received considerable attention because of its utility in the diagnosis and monitoring of pathological conditions. Exogenous and endogenous amino acids are hub substances in regulated metabolic networks, and are also relatively abundant in many biological samples. Therefore, they are thought to have superior properties as targets of focused metabolomics. In several studies, alterations in the free amino acid profile were found to be related to various disease states, such as cancer. 13,14) Previously, we evaluated the effect of several anticancer agents on cancer cell proliferation using amino acid metabolomics analysis of the culture medium. Amino acid measurement was performed using fluorescence der...

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