In the present study, 1 H HRMAS NMR spectroscopy was used to assess the changes in the intracellular metabolic profile of MG-63 human osteosarcoma (OS) cells induced by the chemotherapy agent cisplatin (CDDP) at different times of exposure. Multivariate analysis was applied to the cells spectra, enabling consistent variation patterns to be detected and drug-specific metabolic effects to be identified. Statistical recoupling of variables (SRV) analysis and spectral integration enabled the most relevant spectral changes to be evaluated, revealing significant time-dependent alterations in lipids, choline-containing compounds, some amino acids, polyalcohols, and nitrogenated bases. The metabolic relevance of these compounds in the response of MG-63 cells to CDDP treatment is discussed.

In this work, (1)H high resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR) spectroscopy was used to characterize the variations in the metabolome (small metabolites and mobile lipids) of A549 human lung cells in response to exposure to the alkylating drug cisplatin. Multivariate analysis and signal integration of spectral data were carried out to unveil exposure-induced effects and follow their time course. Parallel and strongly correlated increases in lipids (particularly unsaturated triglycerides) and nucleotide sugars (particularly uridine diphosphate N-acetylglucosamine) were found in cisplatin-treated cells, highlighting these compounds as potential biomarkers of treatment response. Other significant changes upon drug exposure comprised an increase in sorbitol and decreases in niacinamide and several amino acids (glutamine, alanine, lysine, methionine, citrulline, phenylalanine and tyrosine). These results show that in vitro NMR metabolomics is a powerful tool for detecting variations in a range of intracellular compounds upon drug exposure, thus offering the possibility of identifying candidate metabolite markers for in vivo monitoring of tumor responsiveness to treatment.

A high resolution magic angle spinning NMR metabolomics study of the effects of doxorubicin (DOX), methotrexate (MTX) and cisplatin (cDDP) on MG-63 cells is presented and unveils the cellular metabolic adaptations to these drugs, often used together in clinical protocols. Although cDDP-treated cells were confirmed to undergo extensive membrane degradation accompanied by increased neutral lipids, DOX-and MTX-treated cells showed no lipids increase and different phospholipid signatures, which suggests that (i) DOX induces significant membrane degradation, decreased membrane synthesis, and apparent inhibition of de novo lipid synthesis, and (ii) MTX induces decreased membrane synthesis, while no membrane disruption or de novo lipid synthesis seem to occur. Nucleotide signatures were in apparent agreement with the different drug action mechanisms, a link having been found between UDPGlcNAc and the active pathways of membrane degradation and energy metabolism, for cDDP and DOX, with a relation to oxidative state and DNA degradation, for cDDP. Correlation studies unveiled drug-specific antioxidative signatures, which pinpointed m-and s-inositols, taurine, glutamate/ glutamine, and possibly creatine as important in glutathione metabolism. These results illustrate the ability of NMR metabolomics to measure cellular responses to different drugs, a first step toward understanding drug synergism and the definition of new biomarkers of drug efficacy.

The aim of this work was to investigate the effects of cell handling and storage on cell integrity and 1 H high resolution magic angle spinning (HRMAS) NMR spectra. Three different cell types have been considered (lung tumoral, amniocytes, and MG-63 osteosarcoma cells) in order for sample-dependent effects to be identified. Cell integrity of fresh cells and cells frozen in cryopreservative solution was ∼70-80%, with the former showing higher membrane degradation, probably enzymatic, as indicated by increased phosphocholine (PC) and/or glycerophosphocholine (GPC). Unprotected freezing (either gradual or snap-freezing) was found to lyse cells completely, similar to mechanical cell lysis. Besides enhanced metabolites visibility, lysed cells showed a different lipid profile compared to intact cells, with increased choline, PC, and GPC and decreased phosphatidylcholine (PTC). Cell lysis has, therefore, a significant effect on cell lipid composition, making handling reproducibility an important issue in lipid analysis. Sample spinning was found to disrupt 5-25% of cells, depending on cell type, and HRMAS was shown to be preferable to solution-state NMR of suspensions or supernatant, giving enhanced information on lipids and comparable resolution for smaller metabolites. Relaxation-and diffusion-edited NMR experiments gave limited information on intact cells, compared to lysed cells. The 1 H HRMAS spectra of the three cell types are compared and discussed.Nuclear magnetic resonance (NMR) spectroscopy has been, in recent years, increasingly employed for the analysis of metabolic processes in biological systems because of its ability to provide rapid detection of many different metabolites present in complex systems such as biofluids, biological tissues, or cells. The analysis of the metabolome of biological systems provides important information on their biochemical phenotypes and on the metabolic changes occurring in response to external stimuli, e.g., drug exposure, disease onset, medication. 1,2The study of cellular metabolism using NMR has been successfully carried out with strong emphasis on cell extracts, either hydrophilic or lipophilic. For instance, acidic extracts, in the presence of ice-cold perchloric acid (PCA) or trichloroacetic acid (TCA), allow polar metabolites to be identified 3,4 as shown for PCA extracts of human colon adenocarcinoma cells 5 and human osteosarcoma cells 6,7 and TCA extracts of human rhabdomyosarcoma cells 8 and human lung cancer cells. 9 Other extraction methods have been used to identify aqueous and lipophilic metabolites, for instance in human colon carcinoma cells, 10 rat astrocyte cells, 11 human prostate cancer cells, 12 and human lung carcinoma cell lines. 13 In addition to the unavoidable selectivity of extraction methods, rendered useful only when the nature of the compounds of interest is known a priori, sample extraction may involve significant loss of particular cellular components, retained in the residual insoluble precipitate and not amenable to study by solution-st...