Cellular diamine levels in cancer chemoprevention: modulation by ibuprofen and membrane plasmalogens
© Wood et al; licensee BioMed Central Ltd. 2011
Received: 25 October 2011
Accepted: 16 November 2011
Published: 16 November 2011
To develop effective strategies in cancer chemoprevention, an increased understanding of endogenous biochemical mediators that block metastatic processes is critically needed. Dietary lipids and non-steroidal anti-inflammatory drugs (NSAIDs) have a published track record of providing protection against gastrointestinal malignancies. In this regard, we examined the effects of membrane plasmalogens and ibuprofen on regulation of cellular levels of diamines, polyamine mediators that are augmented in cancer cells. For these studies we utilized Chinese hamster ovary (CHO) cells and NRel-4 cells, a CHO cell line with defective plasmalogen synthesis.
NRel-4 cells, which possess cellular plasmalogen levels that are 10% of control CHO cells, demonstrated 2- to 3-fold increases in cellular diamine levels. These diamine levels were normalized by plasmalogen replacement and significantly reduced by ibuprofen. In both cases the mechanism of action appears to mainly involve increased diamine efflux via the diamine exporter. The actions of ibuprofen were not stereospecific, supporting previous studies that cyclooxygenase (COX) inhibition is unlikely to be involved in the ability of NSAIDs to reduce intracellular diamine levels.
Our data demonstrate that ibuprofen, a drug known to reduce the risk of colorectal cancer, reduces cellular diamine levels via augmentation of diamine efflux. Similarly, augmentation of membrane plasmalogens can increase diamine export from control and plasmalogen-deficient cells. These data support the concept that membrane transporter function may be a therapeutic point of intervention for dietary and pharmacological approaches to cancer chemoprevention.
While the focus of cancer research has largely involved the design of cytotoxic molecules, increasing efforts are being made to understand and utilize endogenous anticancer mechanisms. Areas of focus include dietary supplementation, identification of endogenous anticancer metabolites [1, 2] and deregulation of polyamine catabolism [3, 4]. Chemoprevention against colorectal [5–9] and prostate [10, 11] neoplasia has been demonstrated with aspirin and some NSAIDs, like ibuprofen and sulindac, as well as with difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor that decreases polyamine biosynthesis.
While aspirin and NSAIDs act to decrease local inflammation via cyclooxygenase (COX) inhibition, they also produce large decreases in the levels of the polyamines spermidine, putrescine and cadaverine, actions that are independent of COX inhibition . Homeostatic regulation of intracellular polyamine and diamine levels is essential for normal cell growth and restriction of hyperplasia and neoplasia. Regulation of diamine levels is achieved by multiple points of physiological regulation. These include: i) the rate limiting enzyme ODC that converts ornithine to putrescine [13, 14]; ii) polyamine metabolism by polyamine oxidase, spermine oxidase and spermine/spermidine acetyltransferase [15, 16]; iii) polyamine uptake ; and iv) diamine export [17, 18].
Complex changes in plasmalogens and fatty acid precursors of plasmalogens have been reported for cancer cells and the plasma of cancer patients . Dietary omega-3 fatty acids, which are utilized in plasmalogen synthesis, are also known to decrease the risk of several cancers [20, 21]. Decrements in plasmalogen levels, alterations in deacylation-reacylation of plasmalogens, and potential alterations in transport of plasmalogens, resulting from increases in scramblase 1, have all been reported to potentially contribute to neoplasia [22, 23]. In this regard, we have studied plasmalogen deficiency in NRel-4 cells [24–26], a CHO cell mutant not expressing dihydroxyacetone-phosphate acyltransferase (EC 22.214.171.124), a peroxisomal enzyme essential for plasmalogen synthesis. These cells possess plasmalogen levels that are 5 to 10% of those measured in control CHO cells . In a targeted metabolomics analysis utilizing four GC-MS panels that assay over 200 metabolic intermediates in amino acid, nucleotide, alcohol, sugar, polyol, fatty acid, and organic acid pathways , we observed that N-Rel cells had large increases in the intracellular diamines putrescine and cadaverine. In this study we report our findings regarding the effects of plasmalogen replacement  and of ibuprofen treatment on cellular levels of diamines, diamine synthesis, and diamine exporter function in CHO  and NRel  cells.
Materials and methods
CHO and NRel-4 cells (generous gift of Dr. R.A. Zoeller, Boston University) were cultured (10 cm2 plates) in DMEM:F12 (Mediatech) supplemented with 10% FBS (Invitrogen) and 1% antibiotic/antimycotic (Invitrogen). Cells were grown at 37°C in a 5% CO2 incubator and treated with ibuprofen or PPI-1011, an ether lipid plasmalogen precursor, in DMSO at 80% confluence. At the conclusion of incubations, the wells were washed twice with cold phosphate buffered saline (PBS) and the plates harvested with versene/Trypleexpress and stored at -80°C until analyzed.
Cells were incubated with DMSO (0.05% final), ibuprofen (10 μM) or PPI-1011 (50 μM) for 48 hr. Next the cells were washed with PBS and incubated in Hank's Balanced Salt Solution (HBSS) containing 700 μM arginine, to support cellular ornithine synthesis, and 15 mM HEPES (pH7.4) for 1.5 hr. The medium was collected, spiked with [2H4]putrescine, dried with a centrifugal evaporator and assayed for released putrescine. Cells were harvested and the released putrescine expressed as a percentage of the total cellular pool of putrescine.
Cells were incubated with 100 μM [13C5]ornithine in HBSS containing 700 μM arginine and 15 mM HEPES (pH7.4) for 30 min. Cells were washed with cold PBS and harvested as described above, prior to measurement of cellular [13C5]ornithine and [13C4]putrescine pools.
Cells were sonicated in 1.2 ml of acetonitrile:MeOH:formic acid (800:200:2.4) containing [2H4]putrescine, [2H4]cadaverine and [2H5]ornithine (Cambridge and CDN Isotopes) as internal standards. For the putrescine synthesis experiments, [2H4]cadaverine was used as the internal standard. The cell lysates were transferred to 1.5 ml microtubes, sonicated and centrifuged at 4°C and 25,000 × g for 30 min. Next, 400 μL of the supernatant were dried in a centrifugal evaporator. To the dried cell extracts and the dried releasates were added 50 μL of pentafluorobenzyl (PFB) bromide solution (50 μL PFB-Br + 950 μL dimethylformamide) and 10 μL of diisopropylamine as catalyst. The samples were heated with shaking at 80°C for 1 hour and then vortexed with 200 μL of hexane/ethyl acetate (3:2). The tubes were then centrifuged at 25,000 × g for 5 min to precipitate salts. The supernatants were transferred to autosampler vials for GC-MS analyses.
The PFB derivatives were analyzed by ammonia NCI-GC-MS via monitoring the [M-181-3(HF)]- anions for putrescine (567), [2H4]putrescine (571), cadaverine (581), and [2H4]cadaverine (585). The [M-181]- anions were monitored for ornithine (671.1), [2H5]ornithine (676.2), lysine (658.1) and [2H4]lysine (689.1). All GC-MS analyses were performed with an Agilent 7890A GC and Agilent 5975C mass analyzer. The GC column was a 30 m HP-5MS (0.25 mm ID; 0.25 μm film).
CHO and NRel cells were grown on glass coverslips (Thermo Scientific) until plates were approximately 50% confluent. Cells were then fixed by flooding coverslips with 4% paraformaldehyde in PBS for 10 minutes. Following two 5 min rinses in PBS the cells were blocked with 3% skim milk in 0.1% triton X100 PBS for 20 minutes. Cells were stained with an anti-SLC3A2 primary antibody (1:50, Santa Cruz Biotechnology) for 2 hours at room temperature. Excess antibody was removed by rinsing twice in PBS before exposing cells to labeled secondary IgG Alexa 594 antibody (1:400, Invitrogen) for 1 hr at room temperature. Coverslips where then rinsed twice in PBS before applying Hoescht 33258 for 10 min to stain all nuclei. Finally, cells were rinsed twice more in PBS before mounting in Prolong (Molecular Probes) and viewed by fluorescence microscopy.
Data are presented as mean ± SEM for 6 to 8 plates. Metabolite levels were expressed on a per mg protein basis. GC-MS analyses were performed using 5 point standard curves (reference standards at 0.2 to 10 times the stable isotope internal standard). Data were analyzed by 1-way ANOVA, followed by the Tukey-Kramer test for multiple comparisons.
CHO and NRel Diamine Levels
Steady-state levels of amino acids and diamines in untreated CHO and N-Rel cells.
Putrescine (nmol/mg protein)
2.06 ± 0.098
6.69 ± 0.74*
Cadaverine (pmol/mg protein)
123.1 ± 5.1
231.4 ± 27.0*
Ornithine (pmol/mg protein)
703.4 ± 73.4
682.0 ± 41.9
Lysine (nmol/mg protein)
15.1 ± 1.85
13.5 ± 0.90
Arginine (nmol/mg protein)
12.2 ± 0.49
11.6 ± 0.64
Cancer chemoprevention strategies  represent a clinical approach to augmenting endogenous cytoprotective mechanisms to prevent cancer development and constitute an alternative approach to the never-ending search for the magic bullet that only kills cancer cells. Areas of focus in cancer chemoprevention currently include metabolomics of endogenous anticancer metabolites [1, 2]; mechanistic studies of the established cancer prevention provided by regular aspirin or NSAID use ; studies of the roles and mechanisms of dietary omega-3 fatty acids in cancer prevention [20, 21]; and down-regulation of polyamine synthesis to block cancer development [3–6, 8, 9, 11, 13].
Our data also are the first to demonstrate modulation of the diamine exporter by docosahexaenoic acid (DHA)-containing ethanolamine plasmalogens. Augmentation of plasmalogens with the ether lipid precursor, PPI-1011 [25, 27] decreased putrescine levels in CHO cells and normalized putrescine levels in NRel-4 cells to CHO cell levels. As with ibuprofen this appears to involve increased putrescine export to control intracellular diamine homeostasis. These data are consistent with previous reports demonstrating that decrements in membrane plasmalogens dramatically alter membrane function via alterations in membrane lipid rafts which in turn leads to deregulation of cholesterol transport [24, 38], muscarinic membrane receptors  and β-adrenergic membrane receptors [40, 41].
In summary, our understanding of the role(s) of polyamines in the chemoprevention of gastrointestinal malignancies continues to grow. Our data demonstrate the importance of the diamine exporter-SAT complex (Figure 6) in maintaining intracellular diamine levels and further show that a number of dietary and pharmaceutical approaches are available to provide protection against gastrointestinal malignancies.
We thank Dr. R.A. Zoeller for his generous gift of the cell lines. TS was supported by a Canadian National Research Council postdoctoral fellowship.
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