Daniel Morvan

Metabolomics by proton nuclear magnetic resonance spectroscopy of the response to chloroethylnitrosourea reveals drug efficacy and tumor adaptive metabolic pathways.

Metabolomics of tumors may allow discovery of tumor biomarkers and metabolic therapeutic targets. Metabolomics by two-dimensional proton high-resolution magic angle spinning nuclear magnetic resonance spectroscopy was applied to investigate metabolite disorders following treatment by chloroethylnitrosourea of murine B16 melanoma (n = 33) and 3LL pulmonary carcinoma (n = 31) in vivo. Treated tumors of both types resumed growth after a delay. Nitrosoureas provoke DNA damage but the metabolic consequences of genotoxic stress are little known yet. Although some differences were observed in the metabolite profile of untreated tumor types, the prominent metabolic features of the response to nitrosourea were common to both. During the growth inhibition phase, there was an accumulation of glucose (more than x10; P < 0.05), glutamine (x3 to 4; P < 0.01), and aspartate (x2 to 5; P < 0.01). This response testified to nucleoside de novo synthesis down-regulation and drug efficacy. However, this phase also involved the increase in alanine (P < 0.001 in B16 melanoma), the decrease in succinate (P < 0.001), and the accumulation of serine-derived metabolites (glycine, phosphoethanolamine, and formate; P < 0.01). This response witnessed the activation of pathways implicated in energy production and resumption of nucleotide de novo synthesis, thus metabolic pathways of DNA repair and adaptation to treatment. During the growth recovery phase, it remained polyunsaturated fatty acid accumulation (x1.5 to 2; P < 0.05) and reduced utilization of glucose compared with glutamine (P < 0.05), a metabolic fingerprint of adaptation. Thus, this study provides the proof of principle that metabolomics of tumor response to an anticancer agent may help discover metabolic pathways of drug efficacy and adaptation to treatment.

Quantitative HRMAS proton total correlation spectroscopy applied to cultured melanoma cells treated by chloroethyl nitrosourea: Demonstration of phospholipid metabolism alterations

Recent NMR spectroscopy developments, such as high‐resolution magic angle spinning (HRMAS) probes and correlation‐enhanced 2D sequences, now allow improved investigations of phospholipid (Plp) metabolism. Using these modalities we previously demonstrated that a mouse‐bearing melanoma tumor responded to chloroethyl nitrosourea (CENU) treatment in vivo by altering its Plp metabolism. The aims of the present study were to investigate whether HRMAS proton total correlation spectroscopy (TOCSY) could be used as a quantitative technique to probe Plp metabolism, and to determine the Plp metabolism response of cultured B16 melanoma cells to CENU treatment in vitro. The exploited TOCSY signals of Plp derivatives arose from scalar coupling among the protons of neighbor methylene groups within base headgroups (choline and ethanolamine). For strongly expressed Plp derivatives, TOCSY signals were compared to saturation recovery signals and demonstrated a linear relationship. HRMAS proton TOCSY was thus used to provide concentrations of Plp derivatives during long‐term follow‐up of CENU‐treated cell cultures. Strong Plp metabolism alteration was observed in treated cultured cells in vitro involving a down‐regulation of phosphocholine, and a dramatic and irreversible increase of phosphoethanolamine. These findings are discussed in relation to previous in vivo data, and to Plp metabolism enzymatic involvement. Magn Reson Med 49:241–248, 2003.

Bystander effects are induced by CENU treatment and associated with altered protein secretory activity of treated tumor cells A relay for chemotherapy

In a previous study, it was reported that secondary untreated melanoma tumors implanted several weeks after and at distance from primary chloroethylnitrosourea (CENU)‐treated tumors underwent differentiation and growth inhibition. To see whether the primary treated tumor released soluble factors that mediated the secondary tumor response, serum transfer experiments were performed in vivo. Administration of serum from CENU‐treated tumor‐bearing donors arrested tumor proliferation, decreased vessel formation and induced tumor metabolite alterations encompassing glutathione decrease and polyunsaturated fatty acid and phosphoethanolamine increase. These changes mimicked secondary tumor phenotype. To reproduce the model in vitro, cell culture supernatant transfer experiments were performed. CENU‐treated cell cultures showed polyploidy and reactive oxygen species (ROS) production. Cell cultures challenged by a conditioned medium of CENU‐treated cells underwent growth inhibition, cytoskeleton disorders, cytokinesis retardation, metabolite alterations, glutathione decrease and phosphoethanolamine increase, without ROS elicitation. Proteomics of CENU‐treated cell conditioned media revealed altered protein secretion activity by CENU‐treated cells. Among de novo secreted proteins, the most expressed were phosphatidylethanolamine‐binding protein (PEBP), cardiovascular heat shock protein (cHsp), Rho‐associated coiled‐coil forming kinase 2 (ROCK) and actin fragments. These proteins testified of cytoskeleton disorders, growth inhibition and metabolite alterations. This article demonstrates the release by CENU‐treated tumors of growth inhibitory differentiation‐inducing soluble factors. These factors mediate remote bystander effects and attest persistent biological activity of residual tumors after chemotherapy.

Methionine-dependence phenotype of tumors : Metabolite profiling in a melanoma model using L-[methyl-13C]methionine and high-resolution magic angle spinning 1H-13C nuclear magnetic resonance spectroscopy

Tumors frequently have abnormal L‐methionine (Met) metabolism, the so‐called Met‐dependence phenotype that refers to the inability to proliferate in the absence of Met. However, the origin of this phenotype is still unknown and may arise from one of several pathways of Met metabolism. To help characterize the metabolic features of Met‐dependent/independent phenotypes, the fate of the methyl carbon of L‐[methyl‐13C]Met was chased in a murine model of malignant melanoma (B16‐F1) in vitro and in vivo. Growth curves under Met restriction showed that melanoma cells in vitro were Met‐independent, whereas implanted melanoma tumors in vivo were Met‐dependent. Label‐assisted high‐resolution magic angle spinning 1H‐13C NMR spectroscopy metabolite profiling showed that, in vitro, creatine and phosphatidylcholine 13C‐enrichments were poor, but S‐adenosyl‐Met and posttranslationally N‐methylated protein signals were strong. In contrast, in vivo, creatine and phosphatidylcholine enrichments were strong but S‐adenosyl‐Met and N‐methylated protein signals were poor. In addition, in vivo, transsulfuration was very efficient, consumed one‐carbon units originating from the methyl carbon of Met, and yielded taurine labeling. From these data, the Met‐dependent/independent phenotypes appear closely related to the source of one‐carbon units. Thus, L‐[methyl‐13C]Met‐assisted NMR spectroscopy metabolite profiling allowed the discrimination between Met‐dependence and Met‐independence and provided novel mechanistic information on their origin. Magn Reson Med, 2006.

Experimental evaluation of nonlinearities of small-sized insertable gradient coils

A phase imaging technique is proposed to map out and quantify gradient nonlinearities of small-sized insertable gradient coils, assuming the whole-body system gradients are highly linear in the domain of interest. The theory is developed and simple equations are derived to allow quantification. It is applied to a 4-loop 18-cm diameter cylindrical gradient coil of optimal design. Experimental gradient nonlinearity maps are obtained for different fields of view. Gradient non-linearities are quantified locally and in regions of interest, showing close agreement with model data.

Response of melanoma tumor phospholipid metabolism to chloroethyle nitrosourea: a high resolution proton NMR spectroscopy study.

Phospholipid metabolism is tightly involved in tumor growth regulation and tumor cell survival. The response of phospholipid metabolism to chloroethyle nitrosourea treatment is investigated in a murine B16 melanoma model. Measurements of phospholipid derivatives are performed on intact tumor tissue samples using one- and two-dimensional proton NMR spectroscopy. During the tumor growth inhibition phase under treatment, tumors overexpress phosphocholine, phosphoethanolamine, glycerophosphocholine and glycerophosphoethanolamine, whereas phosphatidylcholine and phosphatidylethanolamine levels are maintained to control levels. During re-growth, which remained quantitatively much below control growth, chloroethyle nitrosourea-treated melanoma tumors overexpress phosphocholine and phosphoethanolamine only. In treated melanoma, phosphatidylcholine levels show an inverse relationship with tumor growth rates. In conclusion, chloroethyle nitrosourea-treated melanoma tumors maintain their phosphatidylcholine levels and exhibit transformed phospholipid metabolism phenotype, by mechanisms that could participate in tumor cell survival.