|Metabolic Insights into Cancer in Cells, Tissues & Biofluids|
Choline Kinase Silencing in Breast Cancer Cells Results
in Compensatory Upregulation of Phosphatidylcholine-Specific
Kristine Glunde1, Noriko Mori1, Tomoyo Takagi1, Serena Cecchetti2, Carlo Ramoni2, Egidio Iorio2, Franca Podo2, Zaver M. Bhujwalla1
1The Johns Hopkins University School of Medicine, Baltimore, USA; 2Istituto Superiore di Sanità, Rome, Italy
Choline kinase (Chk) overexpression contributes to the elevated phosphocholine (PCho) and total choline (tCho) levels in breast cancers, detected by MRS. RNA interference-mediated Chk silencing decreased proliferation, increased differentiation, and increased the effect of 5-fluorouracil treatment in breast cancer cells, suggesting its use in anticancer therapy. Here we have shown that cellular PCho, which remained slightly elevated in breast cancer cells in spite of efficient Chk silencing, was possibly due to upregulation of phosphatidylcholine-specific phospholipase C protein expression in these cells. It may therefore be necessary to inhibit such compensatory enzymes along with Chk to achieve sufficient cell kill.
Phosphatidylcholine-Specific Phospholipase C
Contributes to the Increase of Phosphocholine in Ovarian Cancer Cells
Egidio Iorio1, Alessandro Ricci1, Maria Elena Pisanu1, Massimo di Vito1, Rossella Canese1, Delia Mezzanzanica2, Silvana Canevari2, Franca Podo1
1Istituto Superiore di Sanità, Roma, Italy; 2Istituto Nazionale Tumori, Milano, Italy
The activity of different enzymes contributing to the phosphatidylcholine (PC) cycle was investigated in human epithelial ovarian cancer cells, in order to identify the biochemical mechanisms responsible for increased phosphocholine (PCho) levels in ovary cancer. We here report that a strong (13- to 17-fold) activation of PC-specific phospholipase C (PC-plc) is associated in these cancer cells with the already known (13- to 20-fold) increase in choline kinase activity. These results suggest that PC-plc may represent a possible novel target of anticancer therapy.
|Monitoring the Microenvironmental Effects
on Choline Metabolism in Human Breast Cancer Cells
Galit Eliyahu1, Talia Harris1, Nimrod Maril1, Raanan Margalit1, Hadassa Degani1
1Weizmann Institute of Science, Rehovot, Israel
Choline metabolites studied in breast cancer xenografts have indicated that microenvironmental conditions affect choline metabolism leading to changes in phosphocholine and glycerophosphocholine levels. Herein, real-time 31P MRS monitoring of choline metabolites in perfused breast cancer cells subjected to microenvironmental changes are presented. Acidosis induced a two fold decrease in phosphocholine level and hypoxia induced ~30% increase in its level. Extract studies indicated significant increase in glycerophosphocholine under acidosis and hypoxia. In addition to modulating choline metabolism by regulating gene expression processes during breast malignant transformation different, mechanisms of action are involved during acidosis and hypoxia as well.
Elevated Choline Kinase Protein Expression and Activity
Correlate with Increased MRS-Detected Phosphocholine Levels in Ovarian
Egidio Iorio1, Kristine Glunde2, Tomoyo Takagi2, Alessandro Ricci1, Maria Elena Pisanu1, Silvana Canevari3, Zaver M. Bhujwalla2, Franca Podo1
1Istituto Superiore di Sanità, Rome, Italy; 2The Johns Hopkins University School of Medicine, Baltimore, USA; 3Fondazione IRCCS Istituto Nazionale dei Tumori, Milano, Italy
Human ovarian carcinoma cell lines exhibit increased phosphocholine (PCho) and total choline (tCho) concentrations compared to normal and immortalized ovarian epithelial cells, as detected by 1H MRS. Here we have shown for the first time that an MRS-detected increase in choline kinase (Chk) activity and intracellular PCho and tCho concentrations in ovarian cancers was, at least partially, due to elevated Chk protein expression levels. These findings indicate that ovarian carcinomas may be susceptible to Chk-targeted anticancer therapies, which can be evaluated and monitored by 1H MRS detection of tCho and PCho.
Phosphocholine Depletion is a Non-Invasive MRS
Biomarker for PI3K Inhibition in Childhood High-Grade Glioma
Nada S. Al-Saffar1, Lynley Marshall2, Laura Elizabeth Jackson1, Chris Jones2, Paul Workman3, Andrew Pearson2, Martin O. Leach1
1The Institute of Cancer Research and The Royal Marsden NHS Trust, Sutton, UK; 2The Institute of Cancer Reasearch, Sutton, UK; 3The Institute of Cancer Research, Sutton, UK
Phosphoinositide 3-kinases (PI3K) play an important role in the development of glioma, hence, PI3K inhibitors may be exceptionally useful in the treatment of these tumors. Using MRS we have investigated biomarker(s) for PI3K inhibition in pediatric high-grade glioma. Inhibition of PI3K signaling in the pediatric high-grade glioma cells SF188 with the novel class 1A PI3K inhibitor PI103 caused a decrease in PC levels detected by MRS. These results show that MRS could provide a biomarker for the non-invasive monitoring of response to novel PI3K-targeted therapeutic drugs in early stage clinical trials of these inhibitors in children with high-grade glioma.
Absence of Phosphatidylcholine (PC) in Bile of
Cholestatic Patients Could Be a Potential Risk Factor for
Cholangiocarcinoma: A 1H MRS Study
Omkar B. Ijare1, Tedros Bezabeh1, Nils Albiin2, Urban Arnelo2, Bo Lindberg2, Ian C.P. Smith1
1National Research Council Institute for Biodiagnostics, Winnipeg, Canada; 2Karolinska Institutet, Huddinge, Stockholm, Sweden
The major lipid components of human bile are bile salts, cholesterol and phosphatidylcholine. Bile salts in bile have both protective and harmful effects on the biliary system. They form vesicles/mixed micelles with phospholipids and cholesterol in the normal physiology, and exercise harmful effects on cholangiocytes in the absence of phosphatidylcholine. 1H MR spectroscopy of human bile revealed the absence of phosphatidylcholine and elevated levels of glycerophosphocholine in some patients with chronic cholestasis. Phosphatidylcholine is an important phospholipid in bile, protecting cholangiocytes from toxic effects of bile salts. Bile devoid of phosphatidylcholine will be richer in the bile salts resulting in reduced micelle formation. The free bile salts in bile may bring about cholangiocellular damage, a possible risk factor for the development of cholangiocarcinoma.
|Characterization of a Hepatoma Cell Line
in a Novel 3D Bioreactor Flow System Using Hyperpolarized 13C MRS
Kayvan R. Keshari1, John Kurhanewicz, Robert Bok, Albert Chen, David Wilson, Rex Jeffries, Mark Van-Criekge, Dan Vigneron, Jeffrey Macdonald
1University of North Carolina, Chapel Hill, Chapel Hill, North Carolina, USA
Studies of hyperpolarized 13C labeled compounds, by way of the DNP method, have been used to investigate metabolic processes associated with the Warburg effect. The purpose of this study was to apply the DNP hyperpolarization method to a cancer cell line that will lead to potentially different metabolic cycles and behavior. For the first time, JM1 (rat hepatoma) cells were cultured in a 3D NMR compatible bioreactor and injected with hyperpolarized 13C1 pyruvate. These cells exhibited metabolism similar to previously described tumor models as well as intermediates associated with gluconeogenesis. This data suggests that is possible to visualize other metabolic processes.
|Metabolic Activity of Diseased and Healthy Prostate
Cells Investigated as Lactate Formation from Hyperpolarized 13C-Pyruvate
Anna Gisselsson1, Mathilde Lerche1
1Imagnia AB, Malmö, Sweden
Anaerobic conversion of pyruvate to lactate is studied by using an in vitro model with four different prostate cell lines. The LDH isoform LDH5 has a high affinity for pyruvate and is highly up-regulated in prostatic carcinoma. The 13C-lactate formation is significantly higher in cancer cells compared to normal prostate cells after adding 13C-pyruvate. Total LDH activity in each respective cell line follows the same pattern as lactate formation. A high 13C-lactate formation after administration of hyperpolarized 13C-pyruvate to cancer cell suspensions correlates a new potential cancer diagnosis method, DNP-MR, to established diagnostic methods, using blood serum and biopsy analysis.
|13C Succinate Catabolism in Breast Cancer Cells is
Sensitive to Extracellular PH and Glucose and Glutamine Levels
Anthony Mancuso1, Stephen J. Kadlecek, Rahim R. Rizi, Craig B. Thompson
1University of Pennsylvania, Philadelphia, Pennsylvania, USA
Succinic acid metabolism by tumors is of interest because it can be hyperpolarized by the parahydrogen method. Transport of succinic acid across the cell membrane is mediated by dicarboxylic acid transporter proteins. Studies with Ehrlich Ascites Tumor Cells have demonstrated that succinate transport and oxidation is highly pH dependent. In addition, the flux of succinate into the TCA cycle will likely be strongly dependent on the availability of other substrates that can be used for cellular energy and biosynthesis. The goal of this work was to examine the effects of pH, glucose, and glutamine on succinate metabolism in a model breast cancer cell line. The results may be helpful for evaluating the suitability of hyperpolarized 13C succinic acid for studying breast tumors.
Metabolic Biomarkers in Blood Plasma of Tumor Bearing
Mice Detected by 1H NMR Spectra
Radka Stoyanova1, István Pelczer2, Paul Hachem1, Qi Zhao3, Truman R. Brown3, Alan Pollack1
1Fox Chase Cancer Center, Philadelphia, Pennsylvania, USA; 2Princeton University, Princeton, New Jersey, USA; 3Columbia University, New York, New York, USA600 1H NMR spectra from plasma from xenograft mouse model of prostate cancer suggest correlation of the succinate amplitude and the presence of the orthotopically grown prostate tumors. There are variations in the lipid content that also seem to correlate with tumor burden and suggest systematic alteration of lipid metabolism. Orthotopic tumors were developed using several LNCaP cell lines. Pattern recognition techniques are applied to identify the spectral patterns related to tumorigenesis.