31P magnetic resonance spectroscopy of endothelial cells grown in three-dimensional matrigel construct as an enabling platform technology: I. The effect of glial cells and valproic acid on phosphometabolite levels.

Very few studies describe endothelial cell (EC) properties under three-dimensional (3D) conditions using (31)P magnetic resonance spectroscopy (MRS). The authors developed a model in which living ECs growing in Matrigel threads (3D conditions) for 5 days are monitored by (31)P MRS, providing the fingerprint of the major EC phosphometabolites. Organic extracts of membranal phospholipids were also analyzed by (31)P MRS. For comparison and as a model for two-dimensional (2D) tissue culture conditions, (31)P MRS spectra of aqueous extracts of EC phosphometabolites grown under 2D conditions were also evaluated. The phosphometabolites fingerprint of the cells cultured under 3D was significantly different from that of ECs maintained under 2D. Moreover, the pattern of phosphometabolites was affected by coculture with C6-glioma cells and upon treatment with valproic acid, which is under clinical investigation as an antioangiogenic anticancer drug. The major effects were modulation of (i) energy metabolism intermediates such as phosphocreatine, (ii) precursors of phospholipids such as phosphomonoesters, and (iii) degradation products of phospholipids such as glycerophosphocholine. This endothelial model will be usefull as an enabling platform technology for tissue engineering.

31P magnetic resonance spectroscopy of endothelial cells grown in three-dimensional matrigel constructs as an enabling platform technology: II. The effect of anti-inflammatory drugs on phosphometabolite levels.

In the accompanying study, the authors presented phosphometabolite patterns of endothelial cells grown under three-dimensional (3D) conditions using (31)P magnetic resonance spectroscopy (MRS). Here the authors describe the effect of nonsteroidal anti-inflammatory drugs (NSAIDs), using this enabling platform technology, which is relevant for evaluating drug effects in tissue-engineered endothelial constructs. Treatment with indomethacin significantly changed the phosphometabolite fingerprint in this endothelial model, by, respectively, increasing (81%) and decreasing (42%) glycerophosphocholine (GPC) and phosphomonoesters (PM). Furthermore, a safer approach using a NSAID prodrug was also demonstrated in this study with a indomethacin phospholipid-derived prodrug (DP-155). Like the parental drug, DP-155 increased and decreased the levels of GPC and PM by 100% and 20%, respectively. These changes represent useful biomarkers to monitor NSAID effects on endothelized tissue-engineered constructs for the purpose of controlling endothelial cell survival and inflammation upon implantation.

Levels of phospholipid metabolites in breast cancer cells treated with antimitotic drugs: a 31P-magnetic resonance spectroscopy study.

Magnetic resonance spectroscopy (MRS) methods have provided valuable information on cancer cell metabolism. In this study, we characterized the 31P-MR spectra of breast cancer cell lines exhibiting differences in hormonal response, estrogen receptors (positive/negative), and metastatic potential. A correlation was made between the cytotoxic effect of antimitotic drugs and changes in cell metabolism pattern. Because most anticancer drugs are more effective on proliferating cells, our study attempted to elucidate the metabolic profile and specific metabolic changes associated with the effect of anticancer drugs on proliferating breast cancer cell lines. Accordingly, for the 31P-MRS experiments, cells were embedded in Matrigel to preserve their proliferation profile and ability to absorb drugs. The MRS studies of untreated cells indicated that the levels of phosphodiesters and uridine diphosphosugar metabolites were significantly higher in estrogen receptor-positive and low metastatic potential cell lines. 31P-MRS observations revealed a correlation between the mode of action of anticancer drugs and the observed changes in cell metabolic profiles. When cells were treated with antimicrotubule drugs (paclitaxel, vincristine, colchicine, nocodazole), but not with methotrexate and doxorubicin, a profound elevation of intracellular glycerophosphorylcholine (GPC) was recorded that was not associated with changes in phospholipid composition of cell membrane. Remarkably, the rate of elevation of intracellular GPC was much faster in cell population synchronized at G2-M compared with the unsynchronized cells. The steady-state level of GPC for paclitaxel-treated cells was reached after approximately 4 h for synchronized cells and after approximately 24 h (approximate duration of one cell cycle) for the unsynchronized ones. These observations may indicate a correlation between microtubule status and cellular phospholipid metabolism. This study demonstrates that 31P-MRS may have diagnostic value for treatment decisions of breast cancer and reveals new aspects of the mechanism of action of antimicrotubule drugs.

Noninvasive real-time monitoring of intracellular cancer cell metabolism and response to lonidamine treatment using diffusion weighted proton magnetic resonance spectroscopy.

We have used diffusion-weighted proton magnetic resonance spectroscopy (DWMRS) to noninvasively selectively observe only the intracellular metabolites of breast cancer and melanoma cell lines in vitro in real time. Breast cancer cell lines representing different stages in breast cancer progression were chosen for study. Intracellular biochemical profiles of six cell lines perfused in alginate beads were obtained. Spectral differences between groups of cell lines, including choline, lactate, and threonine peaks, were investigated. We also monitored response to the antineoplastic agent, lonidamine (LND), as a function of time and drug concentration in perfused cancer cells. Previous studies reported that this drug induced intracellular acidification and lactate accumulation. Diffusion weighted proton spectra demonstrated a 2- to 9-fold increase in the intracellular lactate signal as a response to LND treatment in several cancer cell lines. These results are consistent with the hypothesis that the principal mechanism of LND in some cancer cells is marked inhibition of lactate transport. Moreover, we have shown that there is a factor of two to three between the response of melanoma cells and that of some types of breast cancer cells. The higher sensitivity of the melanoma cells, as predicted by proton DWMRS, was correlated with changes in water-suppressed magnetic resonance spectra and confirmed by a biological assay. This study demonstrates the feasibility of using DWMRS for monitoring intracellular metabolism and for studying the effects and mechanisms of action of anticancer drugs. We believe that this method can be used for noninvasive clinical applications, such as the differentiation between benign and malignant tissue, real-time monitoring of response to therapy, dose response, and toxicity effects.

Resolution of enantiomers of uridine analogs, potential antiviral agents.

Racemic mixtures of 5-substituted carbocyclic analogs of uracil nucleosides, 2"- and 3"-furyl, 2"- and 3"-thienyl and 2"-selenienyl, potentially anti-viral agents, were resolved using amylose tris(3,5-dimethylphenyl)carbamate as the stationary phase. The mobile phase was n-hexane with ethanol or 2-propanol. Effects of some structural features on the extent of discrimination between the enantiomers were examined through the selectivity and resolution factors as well as the elution order. The structural features were as follows; the type and position of the hetero-atom O, S or Se, in the cyclopentadienyl substituent 5 of the uracil and hydroxymethyl vs. acetoxymethyl groups on the cyclopentene moiety of the uridine analogs. It appeared that effects of the structural features on the separation were solvent dependent, with some very unusual solvent effects. For example, average retention, which did not follow the polarity of the mobile phase modifiers, was much higher when ethanol was used compared to 2-propanol. Also, the elution order of the two enantiomers of several pairs was reversed when ethanol was changed to 2-propanol. In general, ethanol affected higher selectivity and resolution of all the enantiomeric pairs.

Structural features affecting chiral discrimination of terpene derivatives on a carbamated amylose stationary phase.

The chiral discrimination of enantiomeric derivatives of alpha-pinene was studied using amylose tris(3,5-dimethylphenylcarbamate) as a chromatographic stationary phase. The effect of structural features of these enantiomeric pairs on their chromatographic resolution was systematically studied to understand further the correlation between these features and chiral discrimination by the carbamated amylose. Structural analysis by molecular mechanics indicated that the conformation of the alpha-pinene skeleton was preserved with substitution in all its derivatives. However, in spite of the rigidity of the molecular backbone of these molecules, their resolution capabilities were different. Apparently, the site of the hydrogen-bonding substituents affected chiral discrimination by the stationary phase rather than conformational changes. Separation was achieved in spite of the fact that none of the members in the series had an aromatic moiety, and therefore pi-pi interactions with the stationary phase were insignificant. Hence it was concluded that the most important interaction of the terpene derivatives with the carbamated amylose was hydrogen bonding.