Impact of the Pd2Spermine Chelate on Osteosarcoma Metabolism: An NMR Metabolomics Study.

A metabolomics study of Pd2Spermine(Spm) on osteosarcoma MG-63 and osteoblastic HOb cells is presented to assess the impact of the potential palladium drug on cell metabolism compared with cisplatin (cDDP). Despite its higher cytotoxicity, Pd2Spm induced lower (and reversible) metabolic impact on MG-63 cells and the absence of apoptosis; conversely, it induced significant deviations in osteoblastic amino acid metabolism. However, when in combination with doxorubicin and methotrexate, Pd2Spm induced strong metabolic deviations on lipids, choline compounds, amino acids, nucleotides, and compounds related to antioxidative mechanisms (e.g., glutathione, inositol, hypoxanthine), similarly to the cDDP cocktail. Synergetic effects included triggering of lipid biosynthesis by Pd2Spm in the presence of doxorubicin (and reinforced by methotrexate) and changes in the glycosylation substrate uridine diphosphate acetylgalactosamine and methionine and serine metabolisms. This work provides promising results related to the impact of Pd2Spm on osteosarcoma cellular metabolism, particularly in drug combination protocols. Lipid metabolism, glycosylation, and amino acid metabolisms emerge as relevant features for targeted studies to further understand a potential anticancer mechanism of combined Pd2Spm.

Metabolic markers of MG-63 osteosarcoma cell line response to doxorubicin and methotrexate treatment: comparison to cisplatin.

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 UDP-GlcNAc 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.

Nuclear magnetic resonance metabolomics of iron deficiency in soybean leaves.

Iron (Fe) deficiency is an important agricultural concern that leads to lower yields and crop quality. A better understanding of the condition at the metabolome level could contribute to the design of strategies to ameliorate Fe-deficiency problems. Fe-sufficient and Fe-deficient soybean leaf extracts and whole leaves were analyzed by liquid (1)H nuclear magnetic resonance (NMR) and high-resolution magic-angle spinning NMR spectroscopy, respectively. Overall, 30 compounds were measurable and identifiable (comprising amino and organic acids, fatty acids, carbohydrates, alcohols, polyphenols, and others), along with 22 additional spin systems (still unassigned). Thus, metabolite differences between treatment conditions could be evaluated for different compound families simultaneously. Statistically relevant metabolite changes upon Fe deficiency included higher levels of alanine, asparagine/aspartate, threonine, valine, GABA, acetate, choline, ethanolamine, hypoxanthine, trigonelline, and polyphenols and lower levels of citrate, malate, ethanol, methanol, chlorogenate, and 3-methyl-2-oxovalerate. The data indicate that the main metabolic impacts of Fe deficiency in soybean include enhanced tricarboxylic acid cycle activity, enhanced activation of oxidative stress protection mechanisms and enhanced amino acid accumulation. Metabolites showing accumulation differences in Fe-starved but visually asymptomatic leaves could serve as biomarkers for early detection of Fe-deficiency stress.

Nuclear magnetic resonance (NMR) study of the effect of cisplatin on the metabolic profile of MG-63 osteosarcoma cells.

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.

Analytical approaches toward successful human cell metabolome studies by NMR spectroscopy.

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 approximately 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.

NMR metabonomics for mammalian cell metabolism studies.

The detailed knowledge of mammalian cell metabolism and its adjustments to different cell properties and perturbations, such as disease and drug exposure, is of enormous value in the deeper understanding of pathological processes and drug mechanisms, as well as in the development of new and improved methods for diagnosis, follow-up of disease progression and treatment response. This review covers recent developments in the use of NMR-based metabonomics to characterize cellular metabolomes and interpret them in terms of metabolic changes taking place in a wide range of situations. The analytical methodology available is briefly presented and the applications developed so far are reviewed. These include differences in cell properties (e.g., drug resistance, cell cycle stage, specific growth conditions and genetic characteristics) and changes induced in response to different perturbations (e.g., disease, drug exposure and irradiation).

Effects of diabetes mellitus, pressure-overload and their association on myocardial structure and function.

BACKGROUND: Structural and functional changes involved in cardiac injury induced by diabetes mellitus, pressure-overload, or both conditions were evaluated. METHODS: Pressure-overload was established by suprarenal aortic banding in rats. Six weeks later, diabetes was induced by streptozotocin (STZ, 65 mg/kg, intraperitoneally), resulting in four groups: SHAM, banded (BA), diabetic (DM), and diabetic-banded (DM-BA). On the 12th week, left ventricular (LV) structure and function were evaluated. LV function was assessed in vivo with pressure-volume catheters and in vitro by papillary muscles' performance at baseline and in response to isoprenaline (ISO, 10(-8) to 10(-5) M). RESULTS: Compared to SHAM, we observed a significant increase of type-B natriuretic peptide (BA = 370 +/- 110%; DM-BA = 580 +/- 210%), LV mass (BA = 36.8 +/- 3.6%; DM-BA = 32.1 +/- 3.1%), cardiomyocyte diameter (BA = 19.5 +/- 2.3%; DM = 14.3 +/- 1.9%; DM-BA = 11.4 +/- 2.0%), fibrosis (BA = 85 +/- 14%; DM = 145 +/- 28%; DM-BA = 155 +/- 14%), advanced glycation end-product (AGE) deposition (DM = 141 +/- 29%; DM-BA = 166 +/- 46%), contraction (tAT: DM = 13.7 +/- 2.4%; DM-BA = 26.3 +/- 7.1%); a delayed relaxation (tHR: DM = 13.8 +/- 2.6%; DM-BA = 25.5 +/- 9.2%) and a decrease of collagen type-I/type-III ratio (DM = -66.1 +/- 4.6%; DM-BA = -51.9 +/- 5.5). In SHAM animals, ISO (10(-5) M) increased 86.5 +/- 26.2% active tension, 105.3 +/- 20.2% dT/dt(max), and 166.8 +/- 29.9% dT/dt(min). Similar effects were observed in BA and DM animals, whereas in DM-BA these inotropic and lusitropic responses were blunted. Moreover, at a similar resting muscle length, ISO decreased passive tension by 12 +/- 3% in SHAM and 11 +/- 3% in BA, indicating an increase in myocardial distensibility, an effect that was absent in both diabetic groups. CONCLUSION: Long-standing pressure-overload increased LV mass, while diabetes promoted AGE and collagen deposition, which might explain the abolition of ISO-induced increased myocardial distensibility. Association of pressure-overload and diabetes completely blunted the inotropic and lusitropic responses to ISO, with no additional structural damages than in pressure-overload or diabetes alone.