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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.

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.

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.

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.

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.

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.

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.

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.

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.

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, USA