The application of13C NMR to the characterization of phospholipid metabolism in cells (original) (raw)
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NMR studies of the lipid metabolism of T47D human breast cancer spheroids
FEBS Letters, 1990
The in vivo 31p NMR spectrum of T47D human breast cancer cells grown as spheroids shows changes in the phosphomonoester lipid precursors as a function of spheroid size. The ratio of phosphorylethanolamine (PE) to phosphorylcholine (PC) was 1.0_ 0.3 for 3-day-old, 150 tan spheroids. This ratio increased to 2.4+0.4 for spheroids 7 days and older and which were at least 300/tm in diameter. To investigate the phosphatidylethanolamine to phosphatidylcholine (PdylE/PdylC) ratio in the membranes, chloroform/methanol extracts of spheroids were performed. The 31p spectrum of these extracts showed no change with spheroid size, namely the PdylE/PdylC ratio was 0.55-0.06 for spheroids of all ages suggesting that membrane composition is strongly regulated at the precursor level.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1991
31p and 13C-NMR were used to determine the kinetics of choline and ethanolamine incorporation in T47D clone 11 human breast cancer cells grown as small (150 Itm) spheroids. Spheroids were perfused inside the spectrometer with 1,2J3C-labeted choline or 1,2-13C-labeled ethanolamine (0.028 raM) a.'.d me builoGp of labeled phosphorylcholine (PC) or phosphorylethanolamine (PE) was monitored. Alternatively the PC and GPC pools were pretaboled with tac and the reduction of label was monitored. 3tp spectra were recorded from which the overall energetic status as well as total pool sizes could be determined. The ATP content was 8 _+ 1 fmoi/cell, and the total PC and PE pool sizes were 16 and 14 fmol/cell, respectively. PC either increased by 5~ over 24 h or remained constant, while PE remained constant in medium without added ethanolamine but increased 2-fold within 30 h in medium containing ethanolamine, indicating a dependence on precursor concentration in the medium. The np and ~C data yielded similar kinetic results: the rate of the enzymes phosphocholine kinase and phosphoethanolamine Idnase were both on the order of 1.0 fmol/ceil per h, and the rate constants for CTP:phosphecheline cytidyltransferase and CTP:phosphoethanolamine kinase were 0.06 h-t for both enzymes. The kinetics of chofine incorporation did not alter in the presence of 0.028 mM ethanolamine indicating that they have non-competing pathways.
Cancer research, 1990
Using 31P nuclear magnetic resonance spectroscopy we have noninvasively observed metabolic control through the cytidine pathways of phosphatidylcholine and phosphatidylethanolamine synthesis in intact actively metabolizing MDA-MB-231 human breast cancer cells. Perfusion with the phospholipid precursors ethanolamine or choline (2 mM) indicates that the cytidylyltransferase enzymes are rate limiting for both pathways. Complete inhibition of choline kinase with ethanolamine allowed the observation of the utilization of phosphocholine by the rate-limiting enzyme choline-phosphate cytidylyltransferase. The rate was dependent on the phosphocholine concentration. Inhibition of glycerophosphorylcholine phosphodiesterase with accumulation of substrate was also observed and allows an estimate of the flux through the degradative pathways. The human lymphoma cell line MOLT-4 was also found to contain high levels of phosphocholine and phosphoethanolamine. The levels of these precursors in the MO...
Cancer Research
Using "I' nuclear magnetic resonance spectroscopy we have noninvasively observed metabolic control through the cytidine pathways of phosphatidylcholine and phosphatidylethanolamine synthesis in intact ac tively metabolizing MDA-MB-231 human breast cancer cells. Perfusion with the phospholipid precursors ethanolamine or choline (2 HIM)indi cates that the cytidylyltransferase enzymes are rate limiting for both pathways. Complete inhibition of choline kinase with ethanolamine al lowed the observation of the utilization of phosphocholine by the ratelimiting enzyme choline-phosphate cytidylyltransferase. The rate was dependent on the phosphocholine concentration. Inhibition of glycerophosphorylcholine phosphodiesterase with accumulation of substrate was also observed and allows an estimate of the flux through the degradative pathways. The human lymphoma cell line MOLT-4 was also found to contain high levels of phosphocholine and phosphoethanolamine. The levels of these precursors in the MOLT-4 line are lowered by 40% after 6 h when perfused with high dose l-/3-D-arabinofuranosylcytosine (Ara-C) (400 Mm) but are unaffected by 2 «imAra-C or dideoxycytidine. High dose Ara-C also resulted in lysis in 8-10 h. However, the MDA-MB-231 cell line which is not sensitive to Ara-C showed no change in its spectrum when perfused with Ara-C. A potential mechanism based on classic phospholipid metabolism for the lytic effect of high dose Ara-C is discussed.
Substantia, 2022
In the past 30 years there has been a significant increase in the number of publications on phospholipid (PL) metabolism, both for the medical purposes of detection and diagnosis of cancer and for the monitoring of the treatment of human cancers. Most of the work has focused on the pathway that produces phosphatidylcholine, the major component of human cell membranes. The trigger for this research was the advent of applications of NMR spectroscopy in vitro and in vivo in the 1980's and observations that most cancer cells and tumors had significant increases in the water-soluble PL precursors and breakdown products. Increased phosphocholine (PC) has been focused on as a marker for cancer using Magnetic Resonance Spectroscopy (MRS) and Positron Emission Tomography (PET). MRS is now used clinically to aid in the diagnosis and severity of some brain tumors; and choline PET is used for the diagnosis and staging of recurrent prostate cancer, paid for by medical insurance companies. Another major area of research starting in the 1990's was the development of specific choline kinase (CK) inhibitors aimed at the isoenzyme CK-a. This isoenzyme is markedly upregulated in cancer cells and unexpectedly was found to have a role in oncogenic transformation independent of its enzyme function.
Tumour phospholipid metabolism
NMR in Biomedicine, 1999
Abnormalities in phospholipid metabolism represent major hallmarks of cancer cells. Changes in the MRS profiles of aqueous precursors and catabolites of phosphatidylcholine (PtdCho) in cancer lesions allow non invasive monitoring of tumor progression and response to conventional and targeted anti-cancer therapies. Advances and limitations of our present understanding of molecular mechanisms underlying these anomalous metabolic profiles will be here discussed in the light of altered expression and activity of enzymes of the PtdCho cycle and links to dysregulated cell signaling pathways responsible for oncogenesis. An overview will also be provided of a) the role of choline metabolites as possible pharmacodynamic biomarkers of targeted therapies and b) current efforts to identify PtdCho cycle enzymes as possible targets for therapy.
Cancer research, 2001
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 ...
Alterations in the homeostasis of phospholipids and cholesterol by antitumor alkylphospholipids
Lipids in Health and Disease, 2010
The alkylphospholipid analog miltefosine (hexadecylphosphocholine) is a membrane-directed antitumoral and antileishmanial drug belonging to the alkylphosphocholines, a group of synthetic antiproliferative agents that are promising candidates in anticancer therapy. A variety of mechanisms have been suggested to explain the actions of these compounds, which can induce apoptosis and/or cell growth arrest. In this review, we focus on recent advances in our understanding of the actions of miltefosine and other alkylphospholipids on the human hepatoma HepG2 cell line, with a special emphasis on lipid metabolism. Results obtained in our laboratory indicate that miltefosine displays cytostatic activity and causes apoptosis in HepG2 cells. Likewise, treatment with miltefosine produces an interference with the biosynthesis of phosphatidylcholine via both CDP-choline and phosphatidylethanolamine methylation. With regard to sphingolipid metabolism, miltefosine hinders the formation of sphingomy...