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[Cancer Research 60, 5204-5213, September 15, 2000]
© 2000 American Association for Cancer Research

Experimental Therapeutics

Pharmacological Inhibition of Phosphatidylcholine Biosynthesis Is Associated with Induction of Phosphatidylinositol Accumulation and Cytolysis of Neoplastic Cell Lines

Robert E. Finney1, Edward Nudelman, Thayer White, Stuart Bursten, Peter Klein, Laura L. Leer, Nu Wang, David Waggoner, Jack W. Singer and Robert A. Lewis

Cell Therapeutics, Inc., Seattle, Washington 98119


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
De novo production of phosphatidic acid (PA) in tumor cells is required for phospholipid biosynthesis and growth of tumor cells. In addition, PA production by phospholipase D has been cited among the effects of certain oncogenes and growth factors. In this report, it has been demonstrated that enhanced phospholipid metabolism through PA in tumor cells can be exploited pharmacologically for development of anticancer agents, such as CT-2584, a cancer chemotherapeutic drug candidate currently in Phase II clinical trials. By inhibiting CTP:choline-phosphate cytidylyltransferase (CT), CT-2584 caused de novo phospholipid biosynthesis via PA to be shunted away from phosphatidylcholine (PC) and into phosphatidylinositol (PI), the latter of which was doubled in a variety of CT-2584-treated tumor cell lines. In contrast, cytotoxic concentrations of cisplatin did not induce accumulation of PI, indicating that PI elevation by CT-2584 was not a general consequence of chemotherapy-induced cell death. Consistent with this mechanism of action, propranolol, an inhibitor of PA phosphohydrolase and phosphatidylcholine biosynthesis, was also cytotoxic to tumor cell lines, induced PI accumulation, and potentiated the activity of CT-2584 in cytotoxicity assays. As expected from biophysical properties of anionic phospholipids on cellular membranes, CT-2584 cytotoxicity was associated with disruption and swelling of endoplasmic reticulum and mitochondria. We conclude that CT-2584 effects a novel mechanism of cytotoxicity to cancer cells, involving a specific modulation of phospholipid metabolism.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Although PA2 is not a major constituent of biological membranes, it is a key intermediate in phospholipid biosynthesis. De novo biosynthesis of PA from glycerol via glycerol phosphate and then LPA is accomplished by condensation of that moiety with a saturated or monounsturated fatty acyl-CoA by the action of a LPA acyl transferase (Fig. 1Citation ; Ref. 1 ). The newly synthesized PA is a precursor for biosynthesis of major constituents of biological membranes that include PC, PE, and PS through DAG as an intermediate, or for synthesis of anionic phospholipids including PI and cardiolipin through CDP-DAG as an intermediate. This large-scale biosynthesis of phospholipids is required for cell proliferation, especially for that in tumor cells. In particular, PC biosynthesis has been implicated in control of cell proliferation (2 , 3) , and inhibition of PC biosynthesis in tumor cell lines has been associated with apoptosis of tumor cells (4 , 5) .



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  		Fig. 1. Biochemical pathways regulating the trafficking of phospholipids through PA. GK, glycerol kinase; GPAT, glycerophosphate acyltransferase; LPAAT, lisophosphatidic acid acyltransferase; LPP, lipid phosphate phosphatase; DK, diacylglycerol kinase; PIS, PI synthase; CK, choline kinase; CT, CTP:choline-phosphate cytidyltransferase; CPT, CDP-choline:1,2-DAG choline phosphotransferase.

 
Smaller quantities of PA can also be generated from PC by the action of PC-specific PLD (PC-PLD; Fig. 1Citation ), and generation of PA by PC-PLD has been associated with neoplastic transformation. In particular, PC-PLD activity was augmented in surgically resected human gastric carcinoma cells and human breast cancer cells, as compared with adjacent noncancerous tissues (6 , 7) , and this enzymatic activity was increased in response to each of a broad class of mitogenic stimulants, including platelet-derived growth factor (8 , 9) , epidermal growth factor (10) , basic fibroblast growth factor (11) , insulin (12 , 13) , and phorbol esters (14) . Mutations in growth factors or growth factor receptors for some of these activators of PC-PLD elicited the unregulated activities of oncogenes (e.g., v-sis, erb-B, and HER2/neu). Furthermore, elevation of PA levels was observed in fibroblasts neoplastically transformed with ras and fps oncogenes (15) , and an increase in PC-PLD activity was correlated with expression of the src oncogene (16) . PA has been shown recently to have targets implicated in neoplastic transformation and growth factor signal transduction pathways. These targets include phospholipase C{gamma} (9 , 17) , Ras-Gap (18, 19, 20) , Raf-1 (21 , 22) , and a protein tyrosine phosphatase (23) . Additionally, some responses connected with the neoplastic phenotype may also be mediated by utilization of PA to produce other intracellular signaling molecules, including DAG and the potent mitogen, LPA (Fig. 1)Citation . Thus, in tumor cells, critical trafficking of phospholipid metabolism through PA is a result not only of de novo biosynthesis but also of extracellular and oncogene-induced processing of other cellular lipids.

In this report, we demonstrate that CT-2584, an anticancer agent currently in Phase II clinical trials, exploits the enhanced trafficking of lipids through PA in tumor cells to selectively alter the phospholipid composition of these cells and to produce a resultant cytotoxicity.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals.
PBS, growth medium, and all cell culture reagents were purchased from Life Technologies, Inc. (Gaithersburg, MD). All phospholipids that were used as standards were purchased from Avanti, Inc. (Alabaster, AL). HP-TLC-HP-HLF silica gel TLC plates were purchased from Analtech, Inc. (Newark, DE). [U-14C]glycerol and [oleoyl-1-14C]DAG were purchased from Amersham Life Science (Elk Grove, IL). 5-[3 H]Cytidine and [methyl-14C]choline were purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO). DL-Propranolol and cisplatin were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell Culture.
MCF-7 (human breast adenocarcinoma cells), NCI-H460 (human large cell lung carcinoma cells), and NCI-H23 (human non-small cell lung carcinoma cells) were obtained from the American Type Culture Collection (ATCC). Human bone marrow stromal (HBMS) cells were prepared from normal human bone marrow transplant donors under IRB-approved protocols (24) . MCF-7, NCI-H460, and NCI-H23 cells were grown in RPMI supplemented with 10% FBS (heat inactivated at 56°C for 30 min), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. HBMS cells were grown in McCoy’s 5A medium supplemented with 12.5% heat inactivated FBS, 12.5% heat inactivated horse serum, 100 units/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 10ng/ml basic fibroblast growth factor, 1% vitamins, 0.4% essential amino acids, 0.04 mM nonessential amino acids, 1 mM sodium pyruvate, 0.075% (w/v) sodium bicarbonate, and 0.36 µg/ml hydrocortisone (25) . All incubations were at 37°C under 5% CO2, 95% air.

Chemical Synthesis of CT-2584.
11-Bromo-1-undecene was prepared from {omega}-undecylenyl alcohol (Aldrich) by treatment with phosphorus tribromide (0.4 mole equivalents) and pyridine (0.2 mole equivalents) in toluene (as solvent) at 4°C, followed by heating to 50°C for an overnight period. Treatment of 11-bromo-1-undecene with 3-chloroperoxybenzoic acid (1.05 mole equivalents) in 1,2-dichloroethane, initially at 10°C, with subsequent warming to room temperature, provided 11-bromo-1,2-epoxyundecane. Sodium theobromine was obtained after treating a solution of theobromine in ethanol with sodium methoxide (1.0 mole equivalent) followed by solvent evaporation. Heating an equimolar mixture of 11-bromo-1, 2-epoxyundecane and sodium theobromine in DMSO at 80°C provided 1-(10,11-epoxyundecyl)-3,7-dimethylxanthine, which, upon further heating with dodecylamine (2 mole equivalents) in DMSO to 100°C, yielded 1-(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine (CT-2584). CT-2584 purity was determined to be 96–99% by 1H NMR, 13C NMR and HPLC analysis. Treatment of CT-2584 with methanesulfonic acid (0.95 mole equivalents) in hot isopropanol, followed by addition of heptane, gave the methanesulfonic acid salt of CT-2584 which was dissolved in a solution of ethanol, deionized water and Tween 80 (70:29.5:0.5) and stored as a 10 mM stock solution at -20°C.

Cytotoxicity Assays.
The LC50 of CT-2584 was evaluated on a panel of 35 human tumor cell lines. Sub-confluent cultures grown in medium per American Type Culture Collection or supplier were treated for 24 h with 1–50 µM CT-2584 for 24 h. The cells were then incubated for an additional 24 h in growth medium lacking drug before determining their viability. Adherent cells were assayed using BCECF, a mixture of 2',7'-bis-(2-carboxyethyl)-5-carboxyfluorescein and 2',7'-bis-(2-carboxyethyl)-6-carboxyfluorescein acetoxymethyl esters per the manufacturer’s instructions (Molecular Probes, Inc., Eugene, OR). For nonadherent cells, CT96 AQ (Promega, Inc., Madison, WI) was added directly to the cells in growth medium (20 µl of dye/100 µl of medium). After 1–4 h of incubation, cell viability was evaluated by measuring absorbance at 490 nm on a model EL 340 plate reader (Bio Tek Instruments, Inc., Winooski, VT).

Additional cell viability assays on MCF-7, NCI-H460, NCI-H23, and HBMS were performed. Subconfluent cultures were incubated with specified concentrations of CT-2584 or vehicle for the indicated times, and viability was determined using 10% Alamar Blue per the manufacturer’s instructions (Trek Diagnostics Systems, Inc., Chicago, IL). Reduced Alamar Blue (a measure of cell viability) was measured at an excitation wavelength of 530 nm for emission at 590 nm using a model Cytofluor 2300/2350 fluorescence plate reader (Millipore, Inc., Bedford, MA). After subtracting background fluorescence, the percentage of viability was determined by dividing fluorescence of test samples by fluorescence of vehicle-treated cells. The concentration of agent causing 50% lethality was defined as the LC50. In some experiments, cell death was also determined using Sytox-Green, as described by the manufacturer (Molecular Probes, Inc., Eugene, OR). Cell death is associated with a compromised cell membrane, leading to an association of Sytox-Green with cellular nucleic acids and, thus, enhanced fluorescence. For this assay, the excitation wavelength was 485 nm for an emission at 530 nm.

CFU-GM Assay.
To determine whether CT-2584 was cytotoxic to murine bone marrow (MBM) progenitor cells, a CFU-GM assay was performed. MBM cells were extruded from femurs with RPMI 1640 containing 10% FBS, using a 20-gauge needle and a 1-ml syringe. The MBM cells were adjusted to a concentration of 1.0 x 106 cells/ml and incubated with CT-2584 for 6 or 8 h at concentrations ranging from 5 to 30 µM. After incubation, the cells were pelleted at 1000 x g for 5 min, washed twice, and counted. Mononuclear cell counts were performed in 2% glacial acetic acid. MBM cells were added to a standard CFU-GM assay (Stem Cell Technologies, Inc., Vancouver, BC) in quadruplicate at 50,000 cells/plate and incubated for 7 days. Spleen conditioned medium (2% final volume) was used as the source of colony stimulating factor. Colonies (>40 cells) were counted and averaged among quadruplicate samples. Average colony counts in treated samples were normalized for comparisons between experiments. Linear curve-fitting software (Delta Graph version 3.5.2) was used to plot relative colony counts as a function of CT-2584 concentration. An IC50 was interpolated across multiple points between 0.0 and 1.0.

Phospholipid Extraction and Partition.
Tumor cell lines and HBMS cells were grown to 70–90% confluency. Incubations with [14C]glycerol (0.04–0.08 µCi/ml), [14C]-DAG (0.08 µCi/ml), and [14C]choline (0.16 µCi/ml) were carried out either prior to, concurrent with, or after addition of CT-2584 to the cell cultures for the defined periods stated in "Results." Subsequent to treatment with test agents, the medium was aspirated, and the cells were washed once with ice-cold PBS and were scraped into cold PBS with a rubber policeman. The harvested cells and cellular fragments were then pelleted at 1500 rpm in a Beckman CS-6 centrifuge (GH 3.8 swinging bucket rotor) over 5 min. After the supernatant was removed, 6 ml of chloroform-methanol (C-M) (2:1, v/v) were added to the cell pellets. The organic cell suspension was vortexed for 3 min, followed by bath sonication for 10 min with occasional vortexing. One ml of 0.2 M KCl/0.2 M H3PO4 was added to the organic sonicates. The contents were capped and vortexed for 3 min and then bath sonicated for 5 min. To obtain phase separation, the tubes were centrifuged at 1000 x g for 10 min. In each tube, a white band of solid material was evident at the interface between the upper and lower phases. The aqueous upper phase was aspirated and discarded. A Pasteur pipette was then inserted past the margin of the solid band into the lower phase to effect a quantitative recovery of the uncontaminated lower phase by aspiration. The lower phase from each tube was evaporated to dryness in a 37°C nitrogen needle evaporator (N-evap), with the addition of ethanol to azeotropically remove water. The dried lipid material was redissolved by vortexing in 0.5 ml C-M (2:1), dried again, and dissolved once more in 100 µl of C-M (2:1) with brief vortexing and sonication.

Multi-One Dimensional TLC and Quantitation of Lipids.
Methods used for separating lipids using multi-one dimensional TLC were modified from those described by White et al. (26) . Twenty-µl portions of extracted lipids were spotted 7 cm from the bottom of TLC plates in 0.5-cm lanes, each separated by 0.7 cm. The lipids were initially resolved using a mobile phase of chloroform:methanol:ammonium hydroxide (65:30:4) for 90 min. This allowed for maximal separation of PC from PI and other polar phospholipids. To detect PC (approximate Rf, 0.15) and other mobile phospholipids, the top half of the plate was sprayed with a 0.05% solution of the lipid-associating dye, primulin, and the positions of all bands of stained lipid were visualized using a handheld UV lamp, UVL-21 (UVP, Inc., San Gabriel, CA). The plate was then scored horizontally with a diamond pen and broken just below the PC band. The resultant lower portion of the plate was dried in an air stream and rotated 180° for the next chromatographic step in chloroform:methanol:acetic acid (glacial):water (70:30:15:6, v/v), which was allowed to advance two-thirds the distance to the top of the plate. The plate was dried again and chromatographed in chloroform:methanol:acetic acid:water (85:12.5:12.5:3, v/v), which was allowed to ascend to the top of the plate. After the plate was dried once more, the final resolution of the lipids was achieved after repeating this latter chromatography procedure, with solvent exposure halted 5 min after it had reached the top of the plate. After drying, each plate was sprayed with primulin and scanned by laser-excited fluorescent detection on a STORM 840 imaging system (Molecular Dynamics, Sunnyvale, CA). The quantity of separated phospholipids on the TLC plates (in nmol) was determined by integration of variable pixel intensities on Imagequant software and extrapolation from standard curves. After analysis of lipid mass by primulin, the plates were exposed to STORM Phosphor Screens for 96 h, and the amount of [14C]glycerol in separated lipids was quantitated with Imagequant. Unless otherwise stated, the amount of 14C associated with individual phospholipids (in pixel units) was normalized to 14C associated with total phospholipids (PC, PE, PI, PS, and PA).

Identity and purity of each separated lipid was determined by comigration with standards and confirmed by electrospray ionization mass spectrometry. The analyzed lipids were quantitatively recovered in single bands and were homogeneous, based upon these analyses.

Analysis of CK, CT, and CPT Activities in Cell-free Extracts.
Subconfluent MCF-7 cells were incubated with vehicle (70% ethanol and 0.5% Tween 80) or 10 µM CT-2584 for 2 h and then rinsed and harvested in an ice-cold 50 mM Tris-HCl (pH 7.5) buffer containing 50 mM NaCl, 1 mM each DTT, EDTA, EGTA, and benzamidine, and 1 µg/ml each pepstatin A, soybean trypsin inhibitor, E64C, and Pefabloc, with or without added CT-2584 (10 µM). The suspended cells were then sonicated with a Branson Sonifer 450 microtip at power setting 1, 99% duty for three times 30 s on ice. [14C]Choline (1.6 µCi, 40 nmol), ATP (5 µmol), CTP (1 µmol), and MgCl2 (5 µmol) were added to 2 mg of cellular material in a final incubation volume of 500 µl. After a 30 min incubation at 37°C, the incubation was quenched by the addition of chloroform:methanol (1:1, v/v) and extracted by the method of Bligh and Dyer (27) . Aliquots of the aqueous and organic phases were separated on silica thin layer plates using methanol:2.4% NaCl:H2O:NH4OH (50:12.5:37.5:5) or CHCl3:ethanol:AA:H2O (75:45:12:6), respectively. Radiolabeled materials in the extracts were identified by comigration with known standards, and radioactivity was quantitated using the STORM 840 imaging system (Molecular Dynamics, Sunnyvale, CA) and Imagequant software.

[3 H]Cytidine Incorporation into CDP Diacylglycerol.
MCF-7 cells were plated into 150 mm culture dishes containing 25 ml of growth medium at a density of 1.5 x 106 cells/dish. After 48 h incubation, (at ~70–80% confluency), the medium was decreased to 20 ml and supplemented with either 10 µM CT-2584 or vehicle for 2 h. [3 H]Cytidine (100 µCi) was then added and the cultures were incubated for an additional 2 h. After washing twice with ice-cold PBS, cells were harvested in cold PBS containing a protease inhibitor cocktail (1 mM benzamidine and 1 mg/ml each Pefabloc, E64, leupeptin, pepstatin A, and soybean trypsin inhibitor). Phospholipids were then extracted according to the method of Bligh and Dyer (27) , except that 2 M KCl containing 0.2 M H3PO4 was used, instead of water, to separate phases. Lipids in the organic extract were separated using multi-one dimensional TLC, as described above, except that the first dimension was run twice in chloroform:methanol:ammonia:water (60:35:4:1), and the second dimension was run once in chloroform:methanol:acetic acid:water (50:28:4:8). Separated lipids were visualized using primulin and scanned with laser-excited fluorescent detection on a STORM 840 imaging system. The area comigrating with CDP-DAG standards was then scraped, and 3 H beta emissions were quantitated by scintillation counting.

Evaluation of LPP, CDS, PC-PLD, DAG Kinase, and PI Synthase Activities.
Crude membranes, isolated by sonication and 100,000 x g centrifugation of ECV304 cells (a T24 variant) overexpressing human LPP-1, LLP-2, LLP-3, or CDP-DAG synthase-1, were used as the source of the stated enzyme activities. PC-PLD activity was partially purified from membranes of SF-9 insect gut epithelial cells overexpressing the enzyme. Cytosol from Jurkat cells, which contains mostly type I DAG kinase (28) , was used as a source of this enzyme activity, and membranes from MCF-7 cells were used in PI synthase assays. All enzyme assays were performed such that no more than 5% of the substrates was consumed during the reaction to insure linearity of the reaction kinetics. Protein fractions were preincubated for 15–30 min at 25°C or 37°C in appropriate buffer containing protease inhibitors and 0–40 µM CT-2584. LPP activity was assayed using 100 µM 1,2-dioleoyl-[33P]-sn-glycerophosphate in Triton micelles or in PC vesicles in the presence of 5 mM N-ethylmaleimide, as described (29) . CDP-DAG synthase activity was assayed using PA:PC vesicles at Kms for CTP and PA, as described (30) . PC-PLD activity was measured in a broken cell assay as described (31) and in intact CT-2584-treated cells using butan-1-ol trapping (32) . DAG kinase activity was evaluated using an N-octyl-ß-D-glucopyranoside-containing assay, with 1,2-dioleoyl-sn-glycerol as the substrate, as described (33) . PI synthase activity was assayed at Kms for CDP-DAG and inositol in a detergent-free assay, as described (34) .

Electron Microscopy.
NCI-H460 cells were plated into 60-mm culture dishes at a density of 176 cells/mm2 and incubated overnight. After treatment with 10 µM CT-2584 or vehicle, the cells were trypsinized and pelleted at 1000 rpm in a Beckman CS-6 centrifuge (GH 3.8 swinging bucket rotor) for 2 min at 4°C. The cell pellet was washed once with 3 ml of ice-cold PBS and once with 3 ml of cold 0.2 M cacodylate solution. The final cell pellet was fixed overnight at 4°C in half-strength Karnovsky’s fixative and postfixed in 1% collidine-buffered osmium tetroxide. After dehydration, cells were embedded in Epon 812. Ultrathin sections were stained using saturating aqueous uranyl acetate and lead tartrate and examined using a JOEL 100 SX transmission electron microscope operating at 80 kV. Electron microscopy was performed by the electron microscopy facility at the Fred Hutchinson Cancer Research Center (Seattle, WA).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cytotoxic Activity of CT-2584.
Because sphingolipids such as ceramide, sphingosine, and sphingosine 1-phosphate are cytotoxic to tumor cell lines in concert with their modulation of phospholipid metabolism, a series of lipid-like compounds with various degrees of similarities to sphingolipids was synthesized. Included were compounds with amino alcohol and dimethylxanthine domains. CT-2584 (Fig. 2)Citation was selected from this compound library for development and is currently in Phase II clinical trials as a cancer chemotherapeutic drug candidate.



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  		Fig. 2. Chemical structure of CT-2584, 1(11-dodecylamino-10-hydroxyundecyl)-3,7-dimethylxanthine.

 
Twenty-four h of continuous exposure to CT-2584 was cytotoxic to 35 human tumor cell lines, including those resistant to multiple drugs, with LC50s of 4.0 ± 2.3 µM (mean ± SD). The panel of tumor cell lines tested included breast, lung, prostate, sarcoma, colon, melanoma, brain, ovarian, and leukemia cells. Nontumorigenic HBMS cells were 3–4-fold less sensitive to CT-2584 cytotoxicity (LC 50, 15.7 ± 3.1 µM) than the panel of human tumor cell lines. Similarly, the colony-forming capacity of murine bone marrow progenitor cells (CFU-GM) was 3–4-fold less sensitive to inhibition by CT-2584 (IC50, 13.9 ± 6.1 µM) than were human tumor cell lines to CT-2584-mediated cytotoxicity.

CT-2584 Decreases PC and Increases PI Accumulation in Tumor Cell Lines.
Cytotoxic concentrations of CT-2584 were evaluated for effects on phospholipid metabolism in MCF-7 and NCI-H460 cells. [14C]Glycerol was allowed to approach a steady state in the cellular phospholipid pools by incubating for 24 h. The tumor cell lines were then treated with specified concentrations of CT-2584 or vehicle, in the continued presence of [14C]glycerol, for the indicated time, and lipids were extracted and separated using multi-one dimensional TLC. TLC plates were sprayed with primulin to stain the separated phospholipids. The relative quantities of the separated phospholipids were determined by measuring both beta emissions from the [14C]radionuclide and the mass by the amount of primulin stain associated with each phospholipid.

[14C]PC decreased and [14C]PI increased in both NCI-H460 cells (Fig. 3, a and b)Citation and MCF-7 cells (Fig. 3d)Citation at cytotoxic concentrations of CT-2584. A concentration-dependent relationship was observed within the tested range, with effects observed at or above 2–3 µM CT-2584. At 15 µM CT-2584, the amount of [14C]PC relative to total phospholipid (PE, PC, PI, PS, and PA) was reduced by 14–15%, and the relative amount of [14C]PI was increased by 8–15%, as compared with levels observed in vehicle-treated cells. [14C]PE, [14C]PS, and [14C]PA did not markedly change over the concentrations tested. The decrease in [14C]PC represented 24–25% of the PC measured in vehicle-treated cells, whereas the increase in [14C]PI was 124–170% over that in vehicle-treated cells. Primulin staining of phospholipids separated by TLC indicated a comparable decrease in mass of PC, representing 18–20% of PC in vehicle-treated cells, and increase in the mass of PI, which was 71–125% over that in vehicle-treated cells (Fig. 3, c and e)Citation . Similar decreases in PC and increases in PI were demonstrated in NCI-H23 human non-small cell lung cancer cells and DU-145 human prostate cancer cells (data not shown).




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  		Fig. 3. Concentration-dependent effect of CT-2584 on phospholipid metabolism in NCI-H460 and MCF-7 cells. NCI-H460 cells (a–c) and MCF-7 cells (d and e) were incubated with [14C]glycerol for 24 h and then with specified concentrations of CT-2584 or vehicle in the continued presence of [14C]glycerol for 4 h. Lipids were extracted, separated by multi-one dimensional TLC, and stained with primulin. Both the amount of 14C associated with phospholipids (a for NCI-H460 cells) and the amount of primulin stain associated with phospholipids were quantitated. Data are presented as the percentage of 14C associated with each lipid entity relative to 14C associated with the sum of the indicated phospholipids (PE, PC, PI, PS and PA; b and d) or as the percentage of primulin stain associated with each lipid entity relative to the sum of the indicated phospholipids (c and e). The average of triplicate determinations (b–e) are represented. Bars, SD.

 
CT-2584 Induces PI Accumulation Prior to Cell Death.
Some changes in phospholipids would be expected to occur after cells have died, because of loss of regulation and compartmentalization of lipid-metabolizing enzymes, and would not have a causal association with cytotoxicity. In contrast, changes in phospholipid composition prior to overt signs of cell death would be compatible with a cause-effect relationship. To test whether changes in PC and PI occurred as a result of cell death or whether these changes occurred prior to cell death, time courses for cytolysis of NCI-H460 and MCF-7 cells were determined and compared with the time courses for changes in phospholipid metabolism. When treated with 10 µM CT-2584, NCI-H460 (LC50, 3.3 ± 0.3 µM) and MCF-7 (LC50, 4.5 ± 0.5 µM) cells remained viable for more than 4 h, as indicated by exclusion of Sytox-Green, which is a measure of plasma membrane integrity (Fig. 4a)Citation . Some cell death was detectable at 6 h, and all cells were killed within 24 h of exposure to the compound. Similar time courses of cell death were obtained using the Alamar Blue viability assay (data not shown). Although death by apoptosis was not evident in NCI-H460 or MCF-7 cells, cytotoxicity in other cell lines such as U937 (monocytic leukemia) was associated with apoptosis, as evidenced by a decrease in DNA content and by analysis of DNA fragmentation (data not shown).



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  		Fig. 4. Time course for cytotoxicity and accumulation of PI in response to CT-2584. NCI-H460 cells (a, {blacksquare}) and MCF-7 cells (a, {square}) were treated with 10 µM CT-2584 or vehicle for the indicated times and assayed for cytotoxicity using Sytox-Green reagent. Data are presented as the percentage of maximal fluorescence, which was ~5-fold over background fluorescence. For phospholipid analysis, MCF-7 cells (b) and NCI-H460 cells (c) were incubated with [14C]glycerol for 24 h and then with 10 µM CT-2584 or vehicle for the specified times in the continued presence of [14C]glycerol. Lipids were extracted and analyzed as in Fig. 3Citation .

 
To evaluate phospholipid metabolism, phospholipids in NCI-H460 and MCF-7 cells were labeled with [14C]gylcerol for 24 h. The cells were then treated with either 10 µM CT-2584 or vehicle for specified times, in the continued presence of [14C]glycerol, and phospholipids were extracted and evaluated. A decrease in [14C]PC and a corresponding increase in [14C]PI were observed within 2 h of treatment with CT-2584 in both NCI-H460 cells (Fig. 4b)Citation and MCF-7 cells (Fig. 4c)Citation . Maximal changes were observed by 8 h, at which time the amount of [14C]PC relative to total phospholipid was reduced by 10–17%, and [14C]PI was increased 9–13%, as compared with levels observed in vehicle-treated cells. The decrease in [14C]PC represented 16–28% of PC measured in vehicle-treated cells, and the increase in [14C]PI was 102–127% over that in vehicle-treated cells. A small change in [14C]PE was observed by 8 h in MCF-7 cells only, whereas little or no change in [14C]PS or [14C]PA was seen in either cell line. Primulin staining of phospholipids separated by TLC indicated a roughly comparable decrease in the mass of PC, representing 15–20% of PC in vehicle-treated cells, and increase in mass of PI, which was 81–96% over that in vehicle-treated cells (data not shown). Therefore, CT-2584 induced quantitative changes in PC and PI mass prior to the earliest demonstration of cell death.

Induced PI Accumulation Is Specific to CT-2584.
To test whether accumulation of PI is specific to CT-2584 treatment or whether other chemotherapeutic agents might induce the same response as a nonspecific event associated with cytotoxicity, the effect of CT-2584 and cisplatin on phospholipids in NCI-H23 (non-small cell lung carcinoma) cells was examined. NCI-H23 cells were selected based upon their sensitivity to both CT-2584 and cisplatin. The LC50 for treatment with CT-2584 was 4 µM, and 8 µM approached the LC100 (Fig. 5a)Citation . The LC50 for cisplatin was 15 µM, and although an LC100 was not achieved at concentrations up to 100 µM, the cytotoxic effect at 37.5 µM was almost maximal at ~80% of the cells (Fig. 5b)Citation . As had been shown for MCF-7 and NCI-H460 cells, NCI-H23 cells treated with 4 µM CT-2584 for 8 h or 10 µM CT-2584 (2.5 times the LC50) for 4 h or 8 h decreased [14C]PC and accumulated [14C]PI (Fig. 5c)Citation . No changes were observed for [14C]PE, [14C]PS, or [14C]PA. In contrast, there were no changes in [14C]PC, [14C]PI, or any of the other phospholipids after treatment with 15 µM cisplatin for 8 h or 37.5 µM (2.5 times the LC50) cisplatin for 4 h or 8 h (Fig. 5d)Citation .



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  		Fig. 5. Effects of CT-2584 and cisplatin on viability and phospholipid metabolism in NCI-H23, human non-small cell lung carcinoma cells. NCI-H23 cells were treated with the indicated concentrations of CT-2584 (a) or cisplatin (b) for 24 h and assayed for viability 24 h later using Alamar Blue. Data are presented as a percentage of vehicle-treated cells. Arrows, concentrations of each drug used for analyses of phospholipid metabolism. For phospholipid analyses, NCI-H23 cells were incubated with [14C]glycerol for 24 h and then with indicated concentrations of CT-2584 (c) or cisplatin (d) in the continued presence of [14C]glycerol for the indicated times (4 or 8 h). Vehicle control represents cells treated with the same solublizing vehicle as used for CT-2584 or cisplatin. Phospholipids were extracted and analyzed as in Fig. 3Citation . Bars, SD.

 
CT-2584 Inhibits de Novo PC Biosynthesis and Induces de Novo PI Biosynthesis.
That the decrease in PC mass and the increase in PI mass are approximately equal in CT-2584-treated cells (see Fig. 3Citation ) suggests that PI is accumulating at the expense of PC. One possibility to account for this observation is that PC serves as a precursor for PI production after treatment with CT-2584 through activation of PC-PLD. However, in both cellular-based assays and cell-free assays, CT-2584 had no effect on PC-PLD activity (data not shown). Alternatively, the data are consistent with inhibition of de novo PC biosynthesis with concurrent induction of de novo PI biosynthesis. To evaluate the effects of CT-2584 on de novo PC and PI biosynthesis, short-term labeling experiments were performed using [14C]glycerol, [14C]DAG, and [14C]choline. MCF-7 cells were treated with 10 µM CT-2584 or vehicle for 2 h. [14C]Gylcerol, [14C]DAG, or [14C]choline was then incubated with cells for 2–4 additional h in the continued presence of CT-2584 or vehicle. Phospholipids were evaluated as described previously, except the [14C]choline incorporation into PC was normalized to total phospholipid mass because choline does not incorporate into PE, PS, PA, or PI.

Pulse-labeled PC was reduced by 82% using [14C]glycerol (Fig. 6a)Citation , by 59% using [14C]DAG (Fig. 6b)Citation , and by 95% using [14C]choline (Fig. 6c)Citation . Pulse-labeled PI was increased by 550% using [14C]glycerol and by 560% using [14C]DAG. As opposed to charged lipids such as phospholipids, DAG is neutral, and its uptake into mammalian cells has been reported by others (35) and confirmed for MCF-7 cells (data not shown). Therefore, CT-2584 inhibits PC biosynthesis and induces PI biosynthesis. Inhibition of PC biosynthesis appears to be distal to DAG biosynthesis, because DAG incorporation into PC was reduced with CT-2584. In addition, because [14C]PI derived from the [14C]DAG precursor increased in CT-2584-treated cells, it appears that CT-2584 is capable of diverting DAG away from PC biosynthesis and into PI biosynthesis.



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  		Fig. 6. Effect of CT-2584 on de novo PC and PI biosynthesis in MCF-7 cells. MCF-7 cells were treated with 10 µM CT-2584 or vehicle for 2 h. Cells were then incubated with [14C]glycerol (a), [14C]DAG (b), or [14C]choline (c) in the continued presence of CT-2584 for 2 additional h. Phospholipids were extracted and analyzed as in Fig. 3Citation , except [14C]choline incorporation into PC was normalized to total phospholipid mass (PE, PE, PI, PS, and PA), as determined by primulin stain. Bars, SD.

 
Effect of CT-2584 on Cell-free Choline Kinase, CT, and CPT Activity.
The final biosynthetic step for formation of PC is a condensation reaction with DAG and CDP-choline (Fig. 1)Citation . Because CT-2584 inhibition of PC biosynthesis appeared to occur distal to DAG production, the effects of CT-2584 on key enzymes involved in production of CDP-choline (referred to as Kennedy pathway enzymes; CK, CT, and CPT; Ref. 36 ) were evaluated. MCF-7 cells were treated with 10 µM CT-2584 or vehicle for 2 h, and crude cell lysates were prepared in the continued presence of 10 µM CT-2584 or vehicle. The cell lysates were then incubated with [14C]choline for 30 min at 37°C, and incorporation into phosphocholine, CDP-choline, and PC was evaluated (Fig. 7)Citation . [14C]Phosphocholine was not decreased in lysates of CT-2584-treated cells as compared with those from vehicle-treated cells, indicating that CK was not a target for CT-2584. A major effect of CT-2584 was found on production of [14C]CDP-phosphocholine, which was decreased by 79% in lysates of CT-2584-treated cells, as compared with those of vehicle-treated cells. Although a very limited amount of [14C]PC was detected from lysates of treated or untreated cells, the degree of inhibition with CT-2584 was similar to that observed for inhibition of CDP-DAG production, suggesting that CT-2584 did not have a major effect on CPT activity.



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  		Fig. 7. Effect of CT-2584 on cell-free [14C]choline incorporation into phosphocholine, CDP-choline, and PC. MCF-7 cells were treated with 10 µM CT-2584 ({blacksquare}) or vehicle ({square}) for 2 h. Cell lysates were then prepared either in the continued presence of 10 µM CT-2584 or vehicle. [14C]Choline incorporation into phosphocholine, CDP-choline, and PC was determined and represented as a percentage of total 14C label in the assay. Bars, SD.

 
CT-2584-induced PI Production Is Associated with Augmented Production of CDP-DAG.
The accumulation of PI at the expense of PC, as induced by CT-2584, could occur either by augmented trafficking of phospholipids through PA and CDP-DAG intermediates, by decreased utilization of PI to form PI-polyphosphates, or by decreased transfer and exchange of PI and PC catalyzed by the PI transfer protein (37 , 38) . To determine whether trafficking of lipids through CDP-DAG was increased after treatment with CT-2584, MCF-7 cells were treated with either 10 µM CT-2584 or vehicle for 2 h. [3 H]Cytidine was then added, and the cultures were incubated for an additional 2 h in the continuous presence of CT-2584 or vehicle.

Incorporation of [3 H]cytidine into CDP-DAG, as quantitated after TLC, increased from 11,354 ± 591 dpm/mg total cell protein in vehicle-treated cells to 45,979 ± 2,636 dpm/mg total cell protein in CT-2584-treated cells. Because the total amount of cell-associated radioactivity in CT-2584-treated cells was severalfold less than in vehicle-treated cells (data not shown), the increase in [3 H]cytidine-labeled CDP-DAG could not be attributable to an increased rate of incorporation of cytidine into cells. Instead, these data would suggest that the 3–4-fold increase in [3 H]cytidine incorporation in CT-2584-treated cells is likely an underestimation of the increased production of CDP-DAG. In contrast to this analysis of [3 H]cytidine-labeled cells, cell-free assays of CDP-DAG synthase activity were unaffected by CT-2584 (data not shown), suggesting that this enzyme was not a direct target for the drug. In a separate cell-free assay for PI synthase activity, CT-2584 was also without effect (data not shown).

Effect of Propranolol on Phospholipid Metabolism and CT-2584 Cytotoxicity.
If augmented production of PA in tumor cells causes them to be more sensitive to CT-2584 than are nontumorigenic cells, then pharmacological agents that further enhance trafficking of phospholipids into PA might also be expected to increase production of PI and enhance CT-2584 cytotoxicity. Independent of its ß-adrenergic antagonist capacity, propranolol has been shown to inhibit both N-ethylmaleimide-sensitive and -insensitive forms of LPP, a class of enzymes that catalyze the hydrolytic release of the phosphate group from PA with the resultant formation of DAG (see Fig. 1Citation ; Ref. 39 ). Therefore, propranolol would be expected to enhance trafficking of PA into metabolites other than DAG, including PI. MCF-7 cells labeled with [14C]glycerol for 24 h were then treated with specified concentrations of propranolol or vehicle, in the continued presence of [14C]glycerol, for 5 h. 14C-Labeled phospholipids were extracted and analyzed (Fig. 8a)Citation . Propranolol induced accumulation of [14C]PI in MCF-7 cells and caused a corresponding decrease in [14C]PC. Measurable increases in [14C]PI were induced with propranolol concentrations as low as 25 µM, without producing effects on [14C]PE, [14C]PS, or [14C]PA. Similar changes were observed by analysis of primulin-stained phospholipids (data not shown). The effects of propranolol on phospholipid metabolism were therefore similar to those induced by CT-2584 (compare Figs. 3Citation and 4Citation to Fig. 8aCitation ). It was, therefore, not surprising to find that propranolol, like CT-2584, was cytotoxic to MCF-7 cells at concentrations that induced accumulation of PI.



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  		Fig. 8. Concentration-dependent effects of propranolol on phospholipid metabolism and CT-2584 cytotoxicity in MCF-7 cells. a, to determine effects on phospholipid metabolism, MCF-7 cells were incubated with [14C]glycerol for 24 h and then with the indicated concentrations of propranolol or vehicle for 5 h. Phospholipids were then extracted and analyzed as in Fig. 3Citation . Concentrations of propranolol that are reproducibly found to have some cytotoxicity are indicated. b, potentiation of CT-2584 cytotoxicity by propranolol. MCF-7 cells were treated with specified concentrations of propranolol for 1 h, followed by the addition of the indicated concentrations of CT-2584 for 10 h. Cells were then washed free of drugs and incubated an additional 14 h in growth medium lacking drugs, and viability was determined using Alamar Blue. Data are presented as a percentage of viability of propranolol-treated (plus vehicle for CT-2584) cells. Viabilities of cells treated with propranolol (and vehicle of CT-2584) in this particular experiment were as follows: 96% viable at 25 µM, 87% viable at 50 µM, 86% viable at 100 µM, and 66% viable at 200 µM propranolol. c, effects of a combination of CT-2584 and propranolol on phospholipid metabolism. MCF-7 cells were incubated with [14C]glycerol for 24 h. Treatments, in the continued presence of [14C]glycerol, included 4 h with 10 µM CT-2584, 5 h with 100 µM propranolol, or 5 h with 100 µM propranolol in combination with 10 µM CT-2584 for the final 4 h. Lipids were extracted and analyzed as described in Fig. 3Citation . Similar effects were observed in multiple experiments. Bars, SD.

 
The combined effects of propranolol and CT-2584 on cytotoxicity were evaluated on MCF-7 cells. The cells were pretreated with the indicated concentrations of propranolol or vehicle for 1 h. CT-2584 or vehicle was then added to cultures for an additional 10 h. The medium containing drugs was then aspirated from the cell cultures and replaced with fresh growth medium lacking drugs for 14 h. Viability was then determined using the Alamar Blue assay and plotted as a percentage of propranolol-treated (plus vehicle of CT-2584) cells (Fig. 8b)Citation . The combined effect of CT-2584 and propranolol on cytotoxicity was greater than the sum of effects attributable to the individual drugs. Therefore, concentrations of propranolol that induced PI accumulation also potentiated CT-2584 cytotoxicity (compare Fig. 8Citation , compare a with b). Because only the S- enantiomer of propranolol is a ß-adrenergic receptor antagonist and both the R- and S- enantiomers of propranolol synergized with CT-2584 in cytotoxicity assays (data not shown), the effect of propranolol on phospholipid metabolism was therefore unrelated to cyclic 3',5'-AMP production.

Effects of CT-2584 and Propranolol on PI Biosynthesis Are Additive.
If cytotoxicity of CT-2584 and propranolol depend upon reduced PC or induced PI production in tumor cell lines, then the potentiation of CT-2584 cytotoxicity by propranolol would be expected to be reflected by either an additive or synergistic effect on PI accumulation. To test this possibility, the phospholipid pool in MCF-7 cells was radiolabeled with [14C]glycerol for 24 h. Propranolol, at 100 µM final concentration, was then added to cultures. After 1-h incubation with propranolol, 10 µM CT-2584 or vehicle was added to the cell cultures, and the phospholipids were extracted 4 h later (Fig. 8c)Citation . Both CT-2584 and propranolol decreased [14C]PC and increased [14C]PI in the tumor cell line, and a combination of both agents had an additive effect on changes in [14C]PC and [14C]PI. Because propanolol evoked phospholipid changes that are similar to those induced by CT-2584, i.e., decreased PC and augmented PI production, it was questioned whether CT-2584 might also inhibit LPP. This activity was, however, unaffected by CT-2584 in separate assays of LPP-1, LPP-2, and LPP-3 (data not shown). Furthermore, CT-2584 had no direct effect on type I DAG kinase (data not shown).

The Cytotoxic Effect of CT-2584 Is Associated with Swelling and Disruption of Mitochondria and the Rough Endoplasmic Reticulum.
Accumulation of anionic phospholipids is expected to cause loss of membrane integrity attributable to decreased lamellar tendency, change or loss of function of intrinsic membrane proteins, or increased likelihood of intracellular membrane fusion, especially in organelles rich in calcium. To determine the effect of CT-2584 on membrane integrity and subcellular structure, NCI-H460 cells were treated with 10 µM CT-2584 or vehicle for periods of from 1 to 10 h, and changes in subcellular morphology were evaluated by electron microscopy. The most striking morphological effects of CT-2584 were marked swelling and loss of structural integrity of the rough endoplasmic reticulum and mitochondria as early as 5 h, as depicted for a 10-h exposure in Fig. 9Citation . In contrast, even at the 10-h time point, the plasma membrane and nuclear membranes of CT-2584-treated cells were morphologically unaltered from their appearance in cells treated with the vehicle for CT-2584.



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  		Fig. 9. Electron micrographs of CT-2584-treated NCI-H460 cells. NCI-H460 cells were treated with vehicle (a) or 10 µM CT-2584 (b and c) for 10 h, harvested, and prepared for transmission electron microscopy at the indicated magnifications. PM, plasma membrane; NM, nuclear membrane; M, mitochondrion; rER, rough endoplasmic reticulum.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CT-2584 was cytotoxic in vitro for a broad panel of tumor cell lines, including those resistant to multiple drugs. In an independent study, CT-2584 was also shown to produce tumor growth delay in human hepatocellular carcinoma cells in severe combined immunodeficient mice. Equivalent antitumor effects were seen with hepatocelluar carcinoma cells that were resistant to multiple drugs on the basis of overexpression of mdr1 (40) . Furthermore, Phase I clinical data suggested antitumor activity against a variety of cancers and indicated that plasma concentrations up to 3–4 µM were repeatedly achieved,3 which is consistent with concentrations presently shown to elicit biochemical events in tumor cells in vitro.

CT-2584 inhibited PC biosynthesis, as measured by incorporation of [14C]glycerol, [14C]DAG, and [14C]choline, and induced PI biosynthesis, as measured by incorporation of [14C]glycerol and [14C]DAG, in tumor cell lines (Fig. 6)Citation . Inhibition of CTP:choline-phosphate CT was determined to be responsible for the effects of CT-2584 on PC biosynthesis (Fig. 7)Citation . That [3 H]cytidine labeling of CDP-DAG in cellular assays increased with CT-2584 treatment suggests that the increased production of PI used CDP-DAG and, by inference, PA intermediates. Moreover, because utilization of [14C]DAG for PI biosynthesis was augmented in CT-2584-treated cells (Fig. 6)Citation , it appears that inhibition of CT may divert DAG away from PC biosynthesis and into PI biosynthesis. That the enzyme activities that mediate phospholipid metabolism between DAG and PI (CDS, PI synthase, LPP, and DAG kinase) were unaffected by CT-2584 (data not shown) is consistent with this interpretation. Somewhat surprisingly, the CT-2584-effected enhancement of phospholipid trafficking through PA and CDP-DAG did not augment production of cardiolipin (data not shown). This suggests either that PI biosynthesis is favored under the experimental conditions, that the effects on PC and PI biosynthesis do not occur in mitochondria, where cardiolipin is synthesized, or that overproduced cardiolipin is rapidly degraded.

Although we have not definitively demonstrated an etiological role of the reduced PC and enhanced PI production by CT-2584 in the death of tumor cells, the accumulated data are thus far consistent with this hypothesis. Notably, inhibition of PC biosynthesis and accumulation of PI occurred at concentrations of CT-2584 that were cytotoxic (Fig. 3)Citation but prior to biochemical evidence of cytotoxicity (Fig. 4)Citation . Cytotoxic concentrations of propranolol, a drug with an enzymatic effect in the same biochemical pathway, also reduced PC and augmented PI production in tumor cell lines. In addition, the effects of CT-2584 and propranolol on PC and PI were additive, and concentrations of propranolol that induced PI accumulation potentiated CT-2584 cytotoxicity (Fig. 8)Citation . Finally, the effect of CT-2584 on PI was not demonstrable at cytotoxic concentrations of an agent (cisplatin) that killed the same cell type (NCI-H23) by a different, defined mechanism (Fig. 6)Citation .

Quantitatively, PC decreased by as much as 25%, and PI doubled with the final mole percentage of PI approaching 20–25% of total major phospholipids (Figs. 3Citation and 4)Citation in CT-2584-treated cells. Thus, the balance between primarily structural phospholipids (PC and PE) and the anionic phospholipid, PI, is dramatically affected by CT-2584. The structure, function, and integrity of biological membranes are primarily dependent upon lipid composition. PC and the sphingolipid, sphingomyelin, appear to play structural roles in stabilizing bilayer (lamellar) structure and extended, high-radius shapes (e.g., plasma membranes), whereas PE promotes the formation of unilamellar inverted phases and membranes with small radii of curvature (e.g., Golgi bodies and exocytotic vesicles; Refs. 41, 42, 43, 44 ). In contrast, anionic phospholipids such as PA, PI, and cardiolipin (Fig. 1)Citation are primarily required for the function of intrinsic membrane proteins in various organelles (45 , 46) and for membrane fusion (47) . Therefore, the lipid composition of cellular organelles must be balanced to maintain structural integrity while, at the same time, accommodating proteins in functional states and regulating membrane fusion. If balance in lipid composition of membranes is not maintained, then the structural integrity and function of organelles and cells would be compromised.

On the basis of the biophysical data obtained from model systems and biological systems (48) , both the massive decrease in PC and increase in PI as produced by CT-2584 would be expected to contribute to a loss of lamellar structure and membrane integrity. This would be attributable to decreased capacity to maintain a lipid bilayer, change or loss of function in intrinsic membrane proteins, or increased likelihood of intracellular membrane fusion (41 , 42 , 47, 48, 49, 50) . The effect of PI would be most dysregulatory in organelles rich in calcium, (e.g., the endoplasmic reticulum and the mitochondrion), because the spherical radii of the PI polar head groups, when aggregated by Ca 2+, would be too small, relative to the spherical radii of the average acyl domains, to maintain ordered geometry. As a result, the presence of Ca2+ would be expected to further decrease the lamellar tendencies of the bilayer, leading to formation of intramembrane micellar subregions and loss of membrane integrity (41 , 42 , 48, 49, 50) . Consistent with this interpretation, decreased production of PC and increased production of PI was associated with disruption of membrane integrity of both mitochondria and endoplasmic reticulum (Fig. 9)Citation .

The attraction of the biochemical approach exemplified by CT-2584 is theoretically appealing. PC biosynthesis through Kennedy pathway intermediates has been associated with proliferation of tumor cell lines (2 , 3) , and PC turnover was augmented in Ras-transformed cells (51) . The association of augmented production of Kennedy pathway intermediates is further exemplified by the observations that oncogenes (ras, src, raf, and mos) induced choline kinase activity in rodent cells, that generation of phosphocholine is essential for the mitogenic action of certain growth factors (52, 53, 54) , and that at least some human tumors contain augmented levels of phosphomonoesters that include phosphocholine and phosphoethanolamine (55, 56, 57, 58, 59) . In addition, intrinsic oncogenic mechanisms (e.g., the presence of oncogenes derived from receptor and non-receptor tyrosine kinases and members of the ras gene family) would also be expected to induce PA production in tumor cells by activation of PC-PLD (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) . By inhibiting CT and PC biosynthesis, such high rates of PA production in tumor cells, either through high rates of phospholipid biosynthesis or turnover (e.g., through PC-PLD), would be expected to yield higher levels of PI biosynthesis in tumor cells than in non-tumor cells after CT-2584 treatment. Hence, tumor cells would be expected to be more sensitive to cytotoxic mechanisms evoked by CT-2584 than would non-tumor cells. And finally, because CT-2584 resides in cellular membranes (data not shown) and does not have to traverse cellular membranes to reach its biological target, it is not likely to be subject to the drug-extruding molecular pumps that are overexpressed in multidrug resistance tumor cells (60 , 61) . Hence, it was not surprising that CT-2584 was active against multidrug-resistant tumor cell lines both in cell culture (as reported herein) and in vivo (40) .

Agents, such as etoposide, camptothecin, farnesol, and chelerythrine (62) and ether lipids such as 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (5) have also been shown to inhibit PC biosynthesis and induce apoptosis, although the effects of these agents on PI biosynthesis have not been described. At least for etopside and camptothecin, it is not clear whether effects on PC biosynthesis significantly contribute to the cytotoxic activities of the agents, because these agents are thought to function primarily by inhibition of topoisomerases (63) . Although ether lipids appear to inhibit both protein kinase C as well as PC biosynthesis, ectopic expression of CTP:phosphocholine cytidyltransferase was capable of abrogating apoptosis induced by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (5) , suggesting that PC biosynthesis was central to its cytotoxic mechanism of action.

We conclude that phospholipid metabolism in tumor cells may represent an opportunity for development of anticancer agents, as exemplified by CT-2584. Because CT-2584 possesses a unique mechanism of action involving reduced PC mass and production of the anionic phospholipid PI, because it is efficacious against tumor cell lines resistant to multiple drugs, and because the drug is well tolerated by patients, we propose that CT-2584 be used as an index compound for using the altered phospholipid dynamics of neoplastic cells to target cancer chemotherapy.


   FOOTNOTES
 
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.

1 To whom requests for reprints should be addressed, at Cell Therapeutics, Inc., 201 Elliott Avenue West, Suite 400, Seattle, WA 98119. Phone: (206) 282-7100: Fax: (206) 284-6206; E-mail: rfinney{at}ctiseattle.com Back

2 The abbreviations used are: PA, phosphatidic acid; LPA, lysophosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; DAG, diacylglycerol; PI, phosphatidylinositol; CDP, cytidine diphosphate diacylglycerol; PLD, phospholipase D; CFU-GM, colony forming unit granulocyte-macrophage; MBM, murine bone marrow; C-M, chloroform:methanol; LPP, lipid phosphate phosphatase; CDS, CDP-DAG synthase; CT, cytidylyltransferase. Back

3 Unpublished observations. Back

Received 11/22/99. Accepted 7/28/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. West J., Tompkins C. K., Balantac N., Nudelman E., Meengs B., White T., Bursten S., Coleman J., Kumar A., Singer J. W., Leung D. W. Cloning and expression of two human lysophosphatidic acid acyltransferase cDNAs that enhance cytokine-induced signaling responses in cells. DNA Cell Biol., 16: 691-701, 1997.[Medline]
  2. Jackowski S. Coordination of membrane phospholipid synthesis with the cell cycle. J. Biol. Chem., 269: 3858-3867, 1994.[Abstract/Free Full Text]
  3. Jackowski S. Cell cycle regulation of membrane phospholipid metabolism. J. Biol. Chem., 271: 20219-20222, 1996.[Abstract/Free Full Text]
  4. Cui Z., Houweling M., Chen M. H., Record M., Chap H., Vance D. E., Terce F. A genetic defect in phosphatidylcholine biosynthesis triggers apoptosis in Chinese hamster ovary cells. J. Biol. Chem., 271: 14668-14671, 1996.[Abstract/Free Full Text]
  5. Baburina I., Jackowski S. Apoptosis triggered by 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine is prevented by increased expression of CTP: phosphocholine cytidylyltransferase. J. Biol. Chem., 273: 2169-2173, 1998.[Abstract/Free Full Text]
  6. Uchida N., Okamura S., Kuwano H. Phospholipase D activity in human gastric carcinoma. Anticancer Res., 19: 671-675, 1999.[Medline]
  7. Uchida N., Okamura S., Nagamachi Y., Yamashita S. Increased phospholipase D activity in human breast cancer. J. Cancer Res. Clin. Oncol., 123: 280-285, 1997.[Medline]
  8. Plevin R., Cook S. J., Palmer S., Wakelam M. J. Multiple sources of sn-1,2-diacylglycerol in platelet-derived-growth-factor-stimulated Swiss 3T3 fibroblasts. Evidence for activation of phosphoinositidase C and phosphatidylcholine-specific phospholipase D. Biochem. J., 279: 559-565, 1991.
  9. Lee Y. H., Kim H. S., Pai J. K., Ryu S. H., Suh P. G. Activation of phospholipase D induced by platelet-derived growth factor is dependent upon the level of phospholipase C-{gamma} 1. J. Biol. Chem., 269: 26842-26847, 1994.[Abstract/Free Full Text]
  10. Yeo E. J., Exton J. H. Stimulation of phospholipase D by epidermal growth factor requires protein kinase C activation in Swiss 3T3 cells. J. Biol. Chem., 270: 3980-3988, 1995.[Abstract/Free Full Text]
  11. Motoike T., Bieger S., Wiegandt H., Unsicker K. Induction of phosphatidic acid by fibroblast growth factor in cultured baby hamster kidney fibroblasts. FEBS Lett., 332: 164-168, 1993.[Medline]
  12. Farese R. V., Konda T. S., Davis J. S., Standaert M. L., Pollet R. J., Cooper D. R. Insulin rapidly increases diacylglycerol by activating de novo phosphatidic acid synthesis. Science (Washington DC), 236: 586-589, 1987.[Abstract/Free Full Text]
  13. Donchenko V., Zannetti A., Baldini P. M. Insulin-stimulated hydrolysis of phosphatidylcholine by phospholipase C and phospholipase D in cultured rat hepatocytes. Biochim. Biophys. Acta, 1222: 492-500, 1994.[Medline]
  14. Mizunuma M., Tanaka S., Kudo R., Kanoh H. Phospholipase D activity of human amnion cells stimulated with phorbol ester and bradykinin. Biochim. Biophys. Acta, 1168: 213-219, 1993.[Medline]
  15. Martin A., Duffy P. A., Liossis C., Gomez-Munoz A., O’Brien L., Stone J. C., Brindley D. N. Increased concentrations of phosphatidate, diacylglycerol and ceramide in ras- and tyrosine kinase (fps)-transformed fibroblasts. Oncogene, 14: 1571-1580, 1997.[Medline]
  16. Song J., Foster D. A. v-Src activates a unique phospholipase D activity that can be distinguished from the phospholipase D activity activated by phorbol esters. Biochem. J., 294: 711-717, 1993.
  17. Jones G. A., Carpenter G. The regulation of phospholipase C-{gamma} 1 by phosphatidic acid. Assessment of kinetic parameters. J. Biol. Chem., 268: 20845-20850, 1993.[Abstract/Free Full Text]
  18. Tsai M. H., Roudebush M., Dobrowolski S., Yu C. L., Gibbs J. B., Stacey D. W. Ras GTPase-activating protein physically associates with mitogenically active phospholipids. Mol. Cell Biol., 11: 2785-2793, 1991.[Abstract/Free Full Text]
  19. Tsai M. H., Hall A., Stacey D. W. Inhibition by phospholipids of the interaction between R-ras, rho, and their GTPase-activating proteins. Mol. Cell Biol., 9: 5260-5264, 1989.[Abstract/Free Full Text]
  20. Tsai M. H., Yu C. L., Wei F. S., Stacey D. W. The effect of GTPase activating protein upon Ras is inhibited by mitogenically responsive lipids. Science (Washington DC), 243: 522-526, 1988.
  21. Ghosh S., Strum J. C., Sciorra V. A., Daniel L., Bell R. M. Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells. J. Biol. Chem., 271: 8472-8480, 1996.[Abstract/Free Full Text]
  22. Rizzo M. A., Shome K., Vasudevan C., Stolz D. B., Sung T. C., Frohman M. A., Watkins S. C., Romero G. Phospholipase D and its product, phosphatidic acid, mediate agonist-dependent raf-1 translocation to the plasma membrane and the activation of the mitogen-activated protein kinase pathway. J. Biol. Chem., 274: 1131-1139, 1999.[Abstract/Free Full Text]
  23. Chalfant C. E., Kishikawa K., Mumby M. C., Kamibayashi C., Bielawska A., Hannun Y. A. Long chain ceramides activate protein phosphatase-1 and protein phosphatase-2A. Activation is stereospecific and regulated by phosphatidic acid. J. Biol. Chem., 274: 20313-20317, 1999.[Abstract/Free Full Text]
  24. Nemunaitis J., Ruillian M., Tompkins C., Andrews D. F., Singer J. W. Stromal cells, growth factors and matrix proteins Gorin N. C. Douay L. eds. . Experimental Hematology Today, : 53-57, Springer-Verlag New York 1989.
  25. Sensebe L., Li J., Lilly M., Crittenden C., Herve P., Charbord P., Singer J. W. Nontransformed colony-derived stromal cell lines from normal human marrows. I. Growth requirement and myelopoiesis supportive ability.. Exp. Hematol., 23: 507-513, 1995.[Medline]
  26. White T., Bursten S., Federighi D., Lewis R. A., Nudelman E. High-resolution separation and quantification of neutral lipid and phospholipid species in mammalian cells and sera by multi-one-dimensional thin-layer chromatography. Anal. Biochem., 258: 109-117, 1998.[Medline]
  27. Bligh E. G., Dyer W. J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37: 911-917, 1959.
  28. Yamada K., Sakane F., Kanoh H. Immunoquantitation of 80 kDa diacylglycerol kinase in pig and human lymphocytes and several other cells. FEBS Lett., 244: 402-406, 1989.[Medline]
  29. Truett A. P., Bocckino S. B., Murray J. J. Regulation of phosphatidic acid phosphohydrolase activity during stimulation of human polymorphonuclear leukocytes. FASEB J., 6: 2720-2725, 1992.[Abstract]
  30. Carman G. M., Kelley M. J. CDPdiacylglycerol synthase from yeast. Methods Enzymol., 209: 242-247, 1992.[Medline]
  31. Brown H. A., Gutowski S., Moomaw C. R., Slaughter C., Sternweis P. C. ADP-ribosylation factor, a small GTP-dependent regulatory protein, stimulates phospholipase D activity. Cell, 75: 1137-1144, 1993.[Medline]
  32. Exton J. H. Signaling through phosphatidylcholine breakdown. J. Biol. Chem., 265: 1-4, 1990.[Abstract/Free Full Text]
  33. Walsh J. P., Suen R., Glomset J. A. Arachidonoyl-diacylglycerol kinase. Specific in vitro inhibition by polyphosphoinositides suggests a mechanism for regulation of phosphatidylinositol biosynthesis. J. Biol. Chem., 270: 28647-28653, 1995.[Abstract/Free Full Text]
  34. Lykidis A., Jackson P. D., Rock C. O., Jackowski S. The role of CDP-diacylglycerol synthetase and phosphatidylinositol synthase activity levels in the regulation of cellular phosphatidylinositol content. J. Biol. Chem., 272: 33402-33409, 1997.[Abstract/Free Full Text]
  35. Florin-Christensen J., Florin-Christensen M., Delfino J. M., Stegmann T., Rasmussen H. Metabolic fate of plasma membrane diacylglycerols in NIH 3T3 fibroblasts. J. Biol. Chem., 267: 14783-14789, 1992.[Abstract/Free Full Text]
  36. Kent C. Eukaryotic phospholipid biosynthesis. Annu. Rev. Biochem., 64: 315-343, 1995.[Medline]
  37. Kearns B. G., Alb J. G. J., Bankaitis V. Phosphatidylinositol transfer proteins: the long and winding road to physiological function. Trends. Cell Biol., 8: 276-282, 1998.[Medline]
  38. Monaco M. E., Alexander R. J., Snoek G. T., Moldover N. H., Wirtz K. W., Walden P. D. Evidence that mammalian phosphatidylinositol transfer protein regulates phosphatidylcholine metabolism. Biochem. J., 335: 175-179, 1998.
  39. Jamal Z., Martin A., Gomez-Munoz A., Brindley D. N. Plasma membrane fractions from rat liver contain a phosphatidate phosphohydrolase distinct from that in the endoplasmic reticulum and cytosol. J. Biol. Chem., 266: 2988-2996, 1991.[Abstract/Free Full Text]
  40. Marucci L., Varticovski L., Arias I. M. Effect of a xanthine analog on human hepatocellular carcinoma cells (Alexander) in culture and in xenografts in SCID mice. Hepatology, 26: 1195-1202, 1997.[Medline]
  41. Gennis R. B. Biomembranes: Molecular Structure and Function36-84, Springer-Verlag New York 1997.
  42. Cullis P. R., Hope M. J., Tilcock C. P. Lipid polymorphism and the roles of lipids in membranes. Chem. Phys. Lipids, 40: 127-144, 1986.[Medline]
  43. DeKruijff B. Polymorphic regulation of membrane lipid composition. Nature (Lond.), 329: 587-588, 1987.[Medline]
  44. Bloom M., Evans E., Mouritsen O. G. Physical properties of the fluid lipid-bilayer component of cell membranes: a perspective. Q. Rev. Biophys., 24: 293-397, 1991.[Medline]
  45. Liscovitch, M., and Chalifa, V. Signal activated phospholipase D. In: Mordechai Liscovitch (ed.), Signal-activated Phospholipases, pp. 31–64. Austin: R. G. Landes Company, 1994.
  46. Fry M., Green D. E. Cardiolipin requirement for electron transfer in complex I and III of the mitochondrial respiratory chain. J. Biol. Chem., 256: 1874-1880, 1981.[Abstract/Free Full Text]
  47. Zimmerberg J., Vogel S. S., Chernomordik L. V. Mechanisms of membrane fusion. Annu. Rev. Biophys. Biomol. Struct., 22: 433-466, 1993.[Medline]
  48. Aguilar L., Ortega-Pierres G., Campos B., Fonseca R., Ibanez M., Wong C., Farfan N., Naciff J. M., Kaetzel M. A., Dedman J. R., Baeza I. Phospholipid membranes form specific nonbilayer molecular arrangements that are antigenic. J. Biol. Chem., 274: 25193-25196, 1999.[Abstract/Free Full Text]
  49. Kinnunen P. K. On the principles of functional ordering in biological membranes. Chem. Phys. Lipids, 57: 375-399, 1991.[Medline]
  50. Marsh D. Lipid-protein interactions and heterogeneous lipid distribution in membranes. Mol. Membr. Biol., 12: 59-64, 1995.[Medline]
  51. Teegarden D., Taparowsky E. J., Kent C. Altered phosphatidylcholine metabolism in C3H10T1/2 cells transfected with the Harvey-ras oncogene. J. Biol. Chem., 265: 6042-6047, 1990.[Abstract/Free Full Text]
  52. Cuadrado A., Carnero A., Dolfi F., Jimenez B., Lacal J. C. Phosphorylcholine: a novel second messenger essential for mitogenic activity of growth factors. Oncogene, 8: 2959-2968, 1993.[Medline]
  53. Jimenez B., del Peso L., Montaner S., Esteve P., Lacal J. C. Generation of phosphorylcholine as an essential event in the activation of Raf-1 and MAP-kinases in growth factors-induced mitogenic stimulation. J. Cell Biochem., 57: 141-149, 1995.[Medline]
  54. Hernandez-Alcoceba R., Saniger L., Campos J., Nunez M. C., Khaless F., Gallo M. A., Espinosa A., Lacal J. C. Choline kinase inhibitors as a novel approach for antiproliferative drug design. Oncogene, 15: 2289-2301, 1997.[Medline]
  55. Daly P. F., Lyon R. C., Faustino P. J., Cohen J. S. Phospholipid metabolism in cancer cells monitored by 31P NMR spectroscopy. J. Biol. Chem., 262: 14875-14878, 1987.[Abstract/Free Full Text]
  56. Merchant T. E., Gierke L. W., Meneses P., Glonek T. 31P magnetic resonance spectroscopic profiles of neoplastic human breast tissues. Cancer Res., 48: 5112-5118, 1988.[Abstract/Free Full Text]
  57. Ruiz-Cabello J., Cohen J. S. Phospholipid metabolites as indicators of cancer cell function. NMR Biomed., 5: 226-233, 1992.[Medline]
  58. de Certaines J. D., Larsen V. A., Podo F., Carpinelli G., Briot O., Henriksen O. In vivo 31P MRS of experimental tumours. NMR Biomed., 6: 345-365, 1993.[Medline]
  59. Smith T. A., Bush C., Jameson C., Titley J. C., Leach M. O., Wilman D. E., McCready V. R. Phospholipid metabolites, prognosis and proliferation in human breast carcinoma. NMR Biomed., 6: 318-323, 1993.[Medline]
  60. Dalton W. S. Mechanisms of drug resistance in hematologic malignancies. Semin. Hematol., 34: 3-8, 1997.[Medline]
  61. Kuwano M., Toh S., Uchiumi T., Takano H., Kohno K., Wada M. Multidrug resistance-associated protein subfamily transporters and drug resistance. Anticancer Drug Des., 14: 123-131, 1999.[Medline]
  62. Anthony M. L., Zhao M., Brindle K. M. Inhibition of phosphatidylcholine biosynthesis following induction of apoptosis in HL-60 cells. J. Biol. Chem., 274: 19686-19692, 1999.[Abstract/Free Full Text]
  63. Kelland L. Platinum complexes Teicher B. A. eds. . Cancer Therapeutics: Experimental and Clinical Agents, : 93-112, Humana Press Totowa, NJ 1997.



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