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Experimental Therapeutics, Molecular Targets, and Chemical Biology |
1 Cancer Research UK Clinical Magnetic Resonance Research Group, The Institute of Cancer Research and The Royal Marsden NHS Foundation Trust; 2 Cancer Research UK Center for Cancer Therapeutics, The Institute of Cancer Research, Sutton, Surrey, United Kingdom; 3 Cancer Research UK Biomedical Magnetic Resonance Research Group, Department of Basic Medical Sciences, St. George's University of London, London, United Kingdom; and 4 Translational Oncology Unit, Department of Molecular and Cellular Biology of Cancer, Instituto de Investigaciones Biomédicas, Madrid, Spain
Requests for reprints: Martin O. Leach, Cancer Research UK Clinical Magnetic Resonance Research Group, The Institute of Cancer Research, The Royal Marsden NHS Foundation Trust, Downs Road, Sutton, Surrey SM2 5PT, United Kingdom. Phone: 44-20-8661-3338; Fax: 44-20-8661-0846; E-mail: martin.leach{at}icr.ac.uk.
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0.05). Inhibition of choline kinase by MN58b resulted in altered phospholipid metabolism both in cultured tumor cells and in vivo. Phosphocholine levels were found to correlate with choline kinase activities. The decrease in phosphocholine, total choline, and phosphomonoesters may have potential as noninvasive pharmacodynamic biomarkers for determining tumor response following treatment with choline kinase inhibitors. (Cancer Res 2006; 66(1): 427-34) |
Introduction |
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Increased activity of choline kinase has been reported to be associated with an enhanced generation of phosphocholine, mostly independent of the rate of net phosphatidylcholine biosynthesis. Growth factors (5, 6), chemical carcinogens (7, 8), and/or ras oncogene transfection (9, 10) have been shown to cause induction of choline kinase activity, leading to an accumulation of phosphocholine. Furthermore, microinjection of phosphocholine into cells resulted in increased mitogenesis (6). Recently, increased choline kinase activity has been shown in human tumor-derived cell lines and in a variety of human tumors compared with their corresponding normal tissues (11, 12). These findings strongly support the role of choline kinase in tumor transformation and carcinogenesis, suggesting choline kinase as a potential target for the development of anticancer drugs.
A novel molecular therapeutic strategy focused on choline kinase inhibition has been developed recently, resulting in the discovery of a group of compounds with an inhibitory activity against choline kinase (13–15). MN58b (1,4-[4-4'-Bis-{[4-(dimethylamine)pyridinium-1-yl] methyl}diphenyl]butane dibromide) exhibits selective inhibition of choline kinase, inhibits proliferation of cancer cells in vitro, and displays therapeutic activity against human tumor xenografts in vivo (14, 16–19).
The rational development of new molecular cancer therapeutics requires the discovery and validation of biomarkers of drug action (20–22). These are valuable because they can provide proof of concept for the intended mechanism of action in phase I clinical studies, as well as aiding dose selection for phase II trials. A recent study has shown that in the majority of phase I trial designs for solid tumor studies of targeted, noncytotoxic agents, traditional end points rather than molecular measures of drug effects are used for selection of the recommended phase II dose (23). The development of surgically noninvasive end points is particularly important because it avoids the need for tumor biopsy.
Magnetic resonance spectroscopy (MRS) is increasingly used for the study of cellular metabolism, both in vivo (small animals and humans) and in vitro (body fluids, tissue cultures, tissue extracts, and isolated tissues). MRS offers several advantages for metabolic studies, being noninvasive and nondestructive, enabling serial measurements of intact tissues. A further advantage of MRS is the identification and quantification of multiple tissue metabolites from a single spectrum. MRS has been applied to the characterization of neoplastic and normal human tissues (24, 25), early detection of therapeutic response (26, 27), drug development (28), and the identification of metabolic changes associated with apoptosis (29, 30). Using MRS, higher levels of phosphomonoesters comprising phosphocholine and phosphoethanolamine were observed in tumors compared with the corresponding normal tissues (31, 32). Other MRS studies showed that progression from the normal to the malignant phenotype is associated with an increase in both phosphocholine and total choline–containing metabolites (33, 34). Furthermore, a MRS study detected an increase in phosphocholine levels in NIH3T3 cells transfected with mutant ras oncogene compared with the parental cell line (35).
To identify a noninvasive and robust biomarker for choline kinase inhibition and potentially tumor response following MN58b treatment, we did comprehensive proton (1H) and phosphorus (31P) MRS studies in human carcinoma models. 1H-MRS and 31P-MRS evaluation of extracts of two human carcinoma cell lines [MDA-MB-231 (breast) and HT29 (colon)] were used to determine whether any MR spectral changes were associated with choline kinase inhibition after MN58b treatment. We have also examined the effect of MN58b on MDA-MB-231 and HT29 xenograft models to show therapeutic efficacy, to determine whether MRS changes observed in the cell studies were reproducible in vivo, and to investigate whether 1H-MRS and 31P-MRS could provide a noninvasive pharmacodynamic biomarker for the therapeutic action of choline kinase inhibitors in clinical trials.
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Materials and Methods |
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-'-Bis(4-piperidine pyridinium-1-yl)P-xilene dibromide; 2.67 mmol/L) dissolved in double deionized water have been previously described (14, 16). Both drugs are produced at Prof. Lacal's laboratories and are not available as commercial compounds. DMEM, FCS, penicillin, and streptomycin were purchased from Life Technologies (Paisley, United Kingdom). Perchloric acid and potassium hydroxide (KOH) were purchased from Merck (Poole, United Kingdom). Hypnorm was purchased from Jansen Pharmaceuticals (Buckinghamshire, United Kingdom), and Hypnovel was purchased from Roche (Welwyn Garden City, United Kingdom). All other chemicals were purchased from Sigma (Poole, United Kingdom). Cell culture and treatment. MDA-MB-231 and HT29 cancer cell lines (American Type Culture Collection, Manassas, VA) were cultured in DMEM supplemented with 10% FCS, 80 units/mL penicillin, and 80 µg/mL streptomycin at 37°C in 5% CO2. Cell growth inhibition (96 hours) for MDA-MB-231 cells with seeding density of 1 x 103 in 200 µL using 96-well plates, was measured by sulforhodamine B assay to assess IC50 (36). MDA-MB-231 cells were treated with MN58b at pharmacologically active concentrations corresponding to 5 x IC50 (6 µmol/L) for 4, 13, 19, 30, and 48 hours or 6 µmol/L ACG20b (an inactive analogue) for 48 hours at 37°C, and HT29 cells were treated with 2.5 µmol/L MN58b or 2.5 µmol/L ACG20b for 48 hours at 37°C. The cells then underwent trypsinization and trypan blue exclusion assay (37). The effect of treatment on cell number was monitored by counting the number of attached cells in a treated flask and comparing that number with the number of attached cells in a control flask.
Cell cycle analysis. Cell cycle analysis of attached and detached control and treated cells was done on cells (1 x 106) fixed in 70% ethanol, treated with 100 µg/mL RNase A in citrate-buffered saline for 30 minutes at 37°C, and stained with 4 µg/mL propidium iodide (38), using an Elite Enhanced System Performance cell sorter (Beckman Coulter, High Wycombe, United Kingdom) at 488 nm. The cytometry data were analyzed using the WinMdi and Cylchred software (University of Wales College of Medicine, Cardiff, United Kingdom).
HT29 and MDA-MB-231 tumor xenograft models. MF-1 nude mice were injected s.c. in the flank with 0.2 mL of a suspension of HT29 human colon carcinoma cells (2.5 x 107/mL) or MDA-MB-231 human breast carcinoma cells (5 x 107/mL) that had been grown as a monolayer in cell culture (37). Tumor size was calculated by measuring the length, width, and depth of each tumor using calipers and by using the following formula: l x w x d x (
/6). Once an appropriate tumor size (approximate volume of 500 mm3) was established, mice were randomly divided into two groups: one group was treated with MN58b in saline at 4 mg/kg i.p. once a day for 5 days, and one group was treated with saline alone following the same regimen. Animals were treated in accordance with local and national ethical requirements and with the United Kingdom Coordinating Committee on Cancer Research Guidelines for the Welfare of Animals in Experimental Neoplasia (39).
In vitro 1H-MRS and 31P-MRS of cell extracts. To obtain an MR spectrum, 1 x 107 to 2 x 107 cells in logarithmic phase were extracted from cell culture, as previously described (30, 40). Briefly, cells were rinsed with ice-cold saline and fixed with 6 mL of ice-cold methanol. Cells were then scraped off the surface of the culture flask, collected into tubes, and vortexed for 30 seconds at room temperature to optimize phospholipid metabolite extraction from the ruptured cells. Chloroform (6 mL) was then added to each tube followed by an equal volume of deionized water. Following phase separation, the solvent in the upper methanol/water phase was removed using a freeze dryer, and the lipid metabolites were recovered by evaporation of chloroform to dryness (30, 40), and samples were stored at –80°C until analysis. Before acquisition of the MRS spectra, the water-soluble metabolites were resuspended in deuterium oxide (D2O) for 1H-MRS or D2O with 10 mmol/L EDTA (pH 8.2) for 31P-MRS, and the lipid metabolites were resuspended in deuteriated chloroform for 1H-MRS. 1H-MRS and 1H-decoupled 31P-MRS spectra were acquired at room temperature on a 500 MHz Bruker spectrometer (Bruker Biospin, Coventry, United Kingdom) using a 30-degree flip angle, a 1-second relaxation delay, spectral width of 100 ppm, and 32 K data points for 31P; a 30-degree flip angle, a 1-second relaxation delay, spectral width of 12 ppm, and 32 K data points for 1H (lipids); and a 90-degree flip angle, a 1-second relaxation delay, spectral width of 12 ppm, 64 K data points, and HDO resonance suppression by presaturation for 1H (aqueous). Metabolite contents were determined by integration and normalized relative to the peak integral of an internal reference [0.15% 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (water fractions) or 0.03% tetramethylsilane (lipid fractions) for 1H-MRS, and methylene diphosphonic acid (70 µL, 2 mmol/L) for 31P-MRS] and corrected for signal intensity saturation and the number of cells extracted per sample.
In vivo 31P-MRS of HT29 and MDA-MB-231 tumor xenografts. Animals were anesthetized with a single i.p. injection of a Hypnovel/Hypnorm/water (1:1:2) mixture as previously described (37). Animals were placed in the bore of a Varian 4.7-T nuclear magnetic resonance (NMR) spectrometer at St. George's University of London, and tumors were positioned in the center of a 12-mm two-turn 1H/31P surface coil. Image-selected in vivo spectroscopy–localized 31P-MR spectra of the tumors were obtained at 37°C, as previously described (41). Briefly, a gradient strength of up to 7.5 x 10–4 T/cm was applied with adiabatic pulses of 800 milliseconds, a 90-degree sincos excitation pulse, and a sech 180 inversion pulse, with a total repetition time of 3 seconds and 600 averages. 31P-MRS of the tumors was carried out before treatment (i.e., day 1) and 4 days after treatment (i.e., day 5). 31P-MR spectra were quantified using the VARiable PROjection program to determine precise chemical shifts and peak integrals as previously described (42). After the final 31P-MRS study, tumors were freeze clamped and stored at –80°C until analysis.
The surface coils used to obtain the 31P-MRS signal from s.c. tumors in vivo were of nonuniform spatial sensitivity; thus, it is not possible to use an internal standard. Thus, the signal intensities observed in the in vivo 31P-MR spectra are expressed as ratios of metabolites.
In vivo 1H-MRS of HT29 tumor xenografts. Anesthetized mice were placed in the MR system as described above. Voxels were selected from scout gradient echo images, and localized shimming yielded line widths of the order of 20 to 30 Hz. The PRESS localization method with water suppression was used to detect choline with a repetition time of 2 seconds, 64 transients, and echo times of 20, 68, 136, 272, and 408 milliseconds. For unsuppressed water, 16 transients were acquired with the same acquisition variables as above except with a lower receiver gain. After the final 1H-MRS study, tumors were freeze clamped and stored at –80°C until analysis.
MRUI software (42) was used for all spectral processing programs, including preprocessing, fitting, and quantification of peak areas of the observed metabolites. Eddy current correction was done by using the water FID to phase correct the metabolite signal point by point (43). This corrects the phase of the spectrum without any subjective bias. The last 100 data points were used to calculate root-mean-square noise and correct for direct current offsets. Using the choline and water peak areas as a function of echo time, choline and water T2 curves were fitted to a single exponential decay function and choline, and water intercepts (M0) and T2s were derived from these fits. The Levenberg-Marquardt algorithm was used for optimization. The choline concentration was calculated using tumor water as a reference assuming tumor tissue water was 80% (44 mol/L; ref. 44) and according to the equation:
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In vitro 31P-MRS of tumor extracts. The freeze-clamped tumors were extracted in 6% perchloric acid, as previously described (45). Neutralized extracts were freeze-dried and reconstituted in 1 mL D2O, and the extracts (0.5 mL) were placed in 5-mm NMR tubes. For 1H-MRS, the water resonance was suppressed by using gated irradiation centered on the water frequency. 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt (50 µL, 5 mmol/L) was added to the samples for chemical shift calibration and quantification. Immediately before the MRS analysis, the pH of the samples was readjusted to 7 with perchloric acid or KOH. For 31P-MRS, which was carried out after the 1H-MRS study, EDTA (50 µL, 60 mmol/L) was added to each sample for chelation of metals ions, and methylene diphosphonic acid (50 µL, 5 mmol/L) was added to each sample for chemical shift calibration and quantification. The extract spectra for both the control and the treated animals were acquired under identical conditions.
Enzymatic assays of choline kinase activity in cell extracts. Following 4, 13, 19, 30, and 48 hours of treatment with 6 µmol/L MN58b, exponentially growing cells from three p60 plates (1 x 106) were lysed in 80 µL of lysis buffer [1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.3 mol/L NaCl, 25 mmol/L HEPES (pH 7.5), 20 mmol/L ß-glycerophosphate, and 0.1% Triton X-100] to a final concentration of 3 µg/µL. From this, 90 µg of total protein were used per point as source of choline kinase in buffer containing 100 mmol/L Tris-HCl (pH 8), 100 mmol/L MgCl2, and 10 mmol/L ATP, as previously described (11).
Statistical analysis. Data are presented as the mean ± SD, and n 
3 unless otherwise specified. For comparison of metabolite concentrations and ratios, unpaired two-tailed Student's standard t tests were used with a P 0.05 considered statistically significant. The Mann-Whitney U test was done for comparison of proportions, with a P 0.05 considered statistically significant. Regression correlation was used to correlate changes in phosphocholine levels (by MRS) and choline kinase activities.
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Results |
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31P-MR spectra of MDA-MB-231 and HT29 cells treated with ACG20b, the inactive analogue, were also investigated to verify that the 31P-MRS detected drop in phosphocholine is due to the inhibitory effect of MN58b on choline kinase. No statistically significant change in phosphocholine levels was observed in MDA-MB-231 (88 ± 11%, P = 0.2) or HT29 (88 ± 10%, P = 0.2) cells following 48 hours of treatment with ACG20b (Table 1; Fig. 1B and C). Changes in phosphocholine concentrations in MDA-MB-231 cells after a time course of treatment with MN58b are summarized in Table 1 and Fig. 1A.
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Phosphocholine is required for the synthesis of phosphatidylcholine. Hence, to find out whether the drop in phosphocholine would cause a similar drop in the phosphatidylcholine levels, 1H-MR spectra of the lipid fractions of control and MN58b-treated MDA-MB-231 and HT29 cells were measured. No significant changes in phosphatidylcholine levels [using the N(CH3)3 peak resonating at 3.32 ppm] were detected following treatment of both cell lines with MN58b (P > 0.4; data not shown). These results are consistent with previous findings using NIH3T3 cells (18) and human primary lymphocytes (19).
Choline kinase activities in cell extracts and correlation to phosphocholine levels measured by MRS. 14C choline labeling was used as a measure of choline kinase activity in MDA-MB-231 cells after a time course treatment with MN58b (Table 1; Fig. 1A). Significant correlation was found between phosphocholine concentrations (measured by MRS) and choline kinase activities (r2 = 0.95, P = 0.0008).
In vivo 1H-MRS and 31P-MRS of HT29 xenografts. In this study, MN58b inhibited tumor growth in HT29 xenografts by 70% (% treated versus control, % T/C), confirming previous findings (16).
In vivo 1H-MR and 31P-MR spectra from a HT29 tumor pre-MN58b and post-MN58b treatment are shown in Fig. 2A to D. Statistically significant decreases in the total choline concentration (P = 0.01) and phosphomonoester/total phosphorus signal ratio (P = 0.05) were also observed post-MN58b treatment (Table 2A). No statistically significant change in total choline concentration was observed in the control (vehicle treated) tumor group. A statistically significant increase in phosphomonoester/total phosphorus signal ratio (P = 0.04) was observed in the control group.
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In vivo 31P-MRS of the MDA-MB-231 tumors showed statistically significant decreases in the phosphomonoester/total phosphorus signal ratio (P = 0.05) following MN58b treatment (Table 2B). No statistically significant change was observed in the control (vehicle treated) tumor group.
In vitro 31P-MRS of HT29 and MDA-MB-231 tumor extracts. In vitro 31P-MRS analysis of extracts of the HT29 and MDA-MB-231 tumor xenografts allows better resolution of the phosphomonoester signal than is possible in vivo. Reduced levels of phosphocholine (P = 0.03 and P = 0.04, respectively) were seen in MN58b-treated tumors (Table 3A), confirming our in vivo 31P-MRS and 1H-MRS findings. No statistically significant change in phosphoethanolamine, glycerophosphoethanolamine, and glycerophosphocholine levels relative to controls was found in either xenograft model (Table 3).
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0.05) and MDA-MB-231 (P 0.05) tumor extracts when compared with vehicle-treated tumor extracts (Table 3B), confirming our in vivo MRS findings. No significant changes in free choline and glycerophosphocholine levels were found following MN58b treatment in either tumor model.
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Discussion |
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MN58b is a potent choline kinase inhibitor (13, 16–19). The mechanism of action and tumor response to MN58b have been assessed using molecular markers and biochemical assays, especially the selective inhibition of choline kinase and consequently depletion of phosphocholine (16–19). However, a noninvasive direct or surrogate marker of target inhibition and potentially of tumor response to this novel treatment would be a valuable aid to the clinical development of choline kinase inhibitors. We investigated MRS for potential markers of drug activity in two human carcinoma cell lines [MDA-MB-231 (breast) and HT29 (colon)] both in cell culture and in xenograft mouse models. A statistically significant drop in phosphocholine levels (phosphomonoester in vivo) has been observed in cultured cells and in solid tumors of both tumor lines following MN58b treatment. In vivo measurement of phosphomonoester includes both phosphocholine and phosphoethanolamine peaks, which could be separated at high field or by using 1H decoupling. No effect on other 31P-MR detectable metabolites was found. These MRS changes, suggesting inhibition of choline kinase, are consistent with the proposed mechanism of action of MN58b (16, 18, 19). Our data are also consistent with a recent study on choline kinase inhibition by small interfering RNA knockdown, where similar MRS results were observed (47). In the breast carcinoma MDA-MB-231 cells, the drop in phosphocholine levels started as early as 4 hours following treatment with MN58b. This was much earlier than the inhibition of cell proliferation and cell cycle arrest, which were only observed following 48 hours of incubation with MN58b. A significant correlation between phosphocholine levels (by MRS) and choline kinase activities (by 14C labeling) following MN58b treatment (r2 = 0.95, P = 0.0008) was also found. This indicated that the drop in phosphocholine levels could potentially be used as an early marker for response to choline kinase inhibition in treated tumors. On the other hand, no statistically significant change in phosphocholine levels or cell number was observed in both cell lines following treatment with the chemically related but inactive analogue ACG20b. Overall, our data suggest that the MRS detected drop in phosphocholine is indeed due to the inhibitory effect of MN58b on choline kinase.
Time-response experiments using MDA-MB-231 cells showed an association between the decrease in phosphocholine levels and the length of exposure to MN58b. At 4 hours, phosphocholine levels dropped by 19%, then an average 50% inhibition was detected from 13 to 48 hours of treatment with MN58b. A similar percentage drop in choline kinase activity (54%) was also observed. 13C-MRS comparison of long-term and short-term labeling with [1,2-13C]choline suggested that breakdown of membrane phosphatidylcholine contributed substantially to the phosphocholine levels in MDA-MB-231 breast cancer cells compared with normal human mammary epithelial cells (HMEC; ref. 48). In HMECs, phosphocholine was primarily generated from the action of choline kinase in the biosynthetic pathway. This could result in a slower response to choline kinase inhibition in MDA-MB-231 cells compared with normal HMECs.
Membrane phospholipid metabolism is a cell cycle–regulated event. Phospholipid turnover is high during the G1 phase, and the accumulation of phospholipid in the S phase correlates with the abrupt cessation of phospholipid turnover at the G1-S boundary (49). Hence, the effect of MN58b treatment on cell cycle distribution and its relation to the phosphocholine depletion observed was investigated. In attached MDA-MB-231 cells, no statistically significant effect on the cell cycle distribution was detected following treatment with MN58b for up to 30 hours. However, at 48 hours, an accumulation of cells in the G1 phase is detected with a drop in the cell population in both the S and G2 phases. The same effect was also observed in HT29 cells following 48 hours of incubation with MN58b. As previously shown (19), MN58b treatment causes inhibition of the de novo synthesis of phosphocholine via choline kinase, resulting in a reduction in the total intracellular pool of phosphocholine, which in turn results in a G1 arrest. Another study has shown that choline deficiency and hence phosphocholine depletion results in the accumulation of fibroblasts in G1 (50).
There are two phosphatidylcholine biosynthesis pathways in higher eukaryotes, the cytidylyl diphosphate-choline pathway and the methylation pathway. The cytidylyl diphosphate/choline pathway in which choline kinase catalyzes the first phosphorylation reaction, yielding phosphocholine from choline, is the primary route for phosphatidylcholine synthesis in mammals (4). Hence, the effect of phosphocholine depletion following MN58b treatment on phosphatidylcholine biosynthesis was investigated using 1H-MRS of the lipid fractions of control and MN58b-treated MDA-MB-231 and HT29 cells. In keeping with previous metabolic analysis in NIH3T3 cells (18) and human primary lymphocytes (19), no statistically significant changes in phosphatidylcholine levels were detected in attached cell population following treatment of either cell line with MN58b. This could be due to the absolute amount of phosphocholine depletion that we are achieving being low relative to the total pool of phosphatidylcholine, which is the major membrane phospholipid. Furthermore, it is possible that an activation of phosphatidylcholine synthesis via the methylation pathway might have occurred to overcome the drop in phosphatidylcholine synthesis via the cytidylyl diphosphate-choline pathway. Although the methylase is usually not expressed in colon and breast cells, tumor cells could express this enzyme (51). However, our previous results, using either glycerol, choline, or serine labeling as probes, suggest that the methylation pathway is not relevant in this process (19). Quantitative 13C labeling MRS experiments using 13C-labeled choline or ethanolamine would further clarify these observations.
In conclusion, our study showed that MRS can be used to detect a decrease in phosphocholine that is associated with the inhibition of choline kinase by MN58b in human colon and breast carcinoma cell lines. The effects were shown in both cultured cells and in human tumor xenografts. Monitoring the pharmacodynamic effects by MRS (using either 31P or 1H) may provide a noninvasive pharmacodynamic marker for choline kinase inhibition and potentially of tumor response in solid tumors in clinical trials with choline kinase inhibitors.
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Acknowledgments |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank J. Titley for help with flow cytometry analyses.
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Footnotes |
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Received 4/17/05. Revised 7/21/05. Accepted 9/16/05.
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), phosphocholine (PC) levels by MRS (solid columns), and choline kinase activity (open columns) in MDA-MB-231 breast carcinoma cells following treatment with MN58b (6 µmol/L) for the indicated times. *, P < 0.04; **, P < 0.001; $, P < 0.00002; ¥, P = 0.0000003 (statistically significant different from the control); two-tailed unpaired t test was used for all comparisons. Columns, mean of three separate experiments unless otherwise stated (
, n = 4); bars, SD. 
, cell no.; 
, PC; 
, pmol PC/µg prot/min. B, in vitro 31P-MRS of an MDA-MB-231 cell extract without treatment (bottom), or following treatment with MN58b (middle), or inactive analogue ACG20b (top). Peak assignments were as follows: phosphocholine (PC), inorganic phosphate (Pi), glycerophosphocholine (GPC), glycerophosphoethanolamine (GPE), nucleoside triphosphate (
-NTP), uridine triphosphate sugars (UDPs). Confirmed in four separate experiments. Representative spectra. C, % (relative to control) phosphocholine levels in MDA-MB-231 breast and HT29 colon carcinoma cells without treatment (solid columns), or following treatment with MN58b (hatched columns), or ACG20b (open columns) for 48 hours. *, P = 0.03; **, P = 0.00001 (statistically significantly different from the control); two-tailed unpaired t test was used for all comparisons. Columns, mean of three separate experiments unless otherwise stated (¥, n = 4); bars, SD. 
, control; 
, MN58b; 
, ACG20b.
