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.
KeywordsCHO NRel-4 ibuprofen plasmalogens omega-3 fatty acids PPI-1011 putrescine cadaverine diamine exporter ornithine decarboxylase 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 184.108.40.206), 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.
- Ritchie SA, Ahiahonu PW, Jayasinghe D, Heath D, Liu J, Lu Y, Jin W, Kavianpour A, Yamazaki Y, Khan AM, Hossain M, Su-Myat KK, Wood PL, Krenitsky K, Takemasa I, Miyake M, Sekimoto M, Monden M, Matsubara H, Nomura F, Goodenowe DB: Reduced levels of hydroxylated, polyunsaturated ultra long-chain fatty acids in the serum of colorectal cancer patients: implications for early screening and detection. BMC Med. 2010, 8: 13- 10.1186/1741-7015-8-13PubMed CentralView ArticlePubMedGoogle Scholar
- Arakaki AK, Mezencev R, Bowen NJ, Huang Y, McDonald JF, Skolnick J: Identification of metabolites with anticancer properties by computational metabolomics. Mol Cancer. 2008, 7: 57- 10.1186/1476-4598-7-57PubMed CentralView ArticlePubMedGoogle Scholar
- Casero RA, Marton LJ: Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases. Nat Rev Drug Discov. 2007, 6: 373-90. 10.1038/nrd2243View ArticlePubMedGoogle Scholar
- Casero RA, Pegg AE: Polyamine catabolism and disease. Biochem J. 2009, 421: 323-38. 10.1042/BJ20090598PubMed CentralView ArticlePubMedGoogle Scholar
- Martinez ME, O'Brien TG, Fultz KE, Babbar N, Yerushalmi H, Qu N, Guo Y, Boorman D, Einspahr J, Alberts DS, Gerner EW: Pronounced reduction in adenoma recurrence associated with aspirin use and a polymorphism in the ornithine decarboxylase gene. Proc Natl Acad Sci USA. 2003, 100: 7859-64. 10.1073/pnas.1332465100PubMed CentralView ArticlePubMedGoogle Scholar
- Thompson PA, Wertheim BC, Zell JA, Chen WP, McLaren CE, LaFleur BJ, Meyskens FL, Gerner EW: Levels of rectal mucosal polyamines and prostaglandin E2 predict ability of DFMO and sulindac to prevent colorectal adenoma. Gastroenterology. 2010, 139: 797-805. 10.1053/j.gastro.2010.06.005PubMed CentralView ArticlePubMedGoogle Scholar
- Harris RE, Beebe-Donk J, Doss H, Burr Doss D: Aspirin, ibuprofen, and other non-steroidal anti-inflammatory drugs in cancer prevention: a critical review of non-selective COX-2 blockade (review). Oncol Rep. 2005, 13: 559-83.PubMedGoogle Scholar
- Meyskens FL, Gerner EW, Emerson S, Pelot D, Durbin T, Doyle K, Lagerberg W: Effect of alpha-difluoromethylornithine on rectal mucosal levels of polyamines in a randomized, double-blinded trial for colon cancer prevention. J Natl Cancer Inst. 1998, 90: 1212-8. 10.1093/jnci/90.16.1212View ArticlePubMedGoogle Scholar
- Zell JA, Pelot D, Chen WP, McLaren CE, Gerner EW, Meyskens FL: Risk of cardiovascular events in a randomized placebo-controlled, double-blind trial of difluoromethylornithine plus sulindac for the prevention of sporadic colorectal adenoma. Cancer Prev Res (Phila). 2009, 2: 209-12. 10.1158/1940-6207.CAPR-08-0203.View ArticleGoogle Scholar
- Mahmud SM, Franco EL, Turner D, Platt RW, Beck P, Skarsgard D, Tonita J, Sharpe C, Aprikian AG: Use of non-steroidal anti-inflammatory drugs and prostate cancer risk: a population-based nested case-control study. PLoS One. 2011, 6: e16412- 10.1371/journal.pone.0016412PubMed CentralView ArticlePubMedGoogle Scholar
- Simoneau AR, Gerner EW, Nagle R, Ziogas A, Fujikawa-Brooks S, Yerushalmi H, Ahlering TE, Lieberman R, McLaren CE, Anton-Culver H, Meyskens FL: The effect of difluoromethylornithine on decreasing prostate size and polyamines in men: results of a year-long phase IIb randomized placebo-controlled chemoprevention trial. Cancer Epidemiol Biomarkers Prev. 2008, 17: 292-9. 10.1158/1055-9965.EPI-07-0658PubMed CentralView ArticlePubMedGoogle Scholar
- Eklou-Kalonji E, Andriamihaja M, Reinaud P, Mayeur C, Camous S, Robert V, Charpigny G, Blachier F: Prostaglandin-independent effects of aspirin on cell cycle and putrescine synthesis in human colon carcinoma cells. Can J Physiol Pharmacol. 2003, 81: 443-50. 10.1139/y03-058View ArticlePubMedGoogle Scholar
- Babbar N, Gerner EW: Targeting polyamines and inflammation for cancer prevention. Recent Results Cancer Res. 2011, 188: 49-64.PubMed CentralView ArticlePubMedGoogle Scholar
- Pegg AE: Regulation of ornithine decarboxylase. J Biol Chem. 2006, 281: 14529-32. 10.1074/jbc.R500031200View ArticlePubMedGoogle Scholar
- Seiler N, Raul F: Polyamines and the intestinal tract. Crit Rev Clin Lab Sci. 2007, 44: 365-411. 10.1080/10408360701250016View ArticlePubMedGoogle Scholar
- Seiler N: Catabolism of polyamines. Amino Acids. 2004, 26: 217-33.PubMedGoogle Scholar
- Uemura T, Gerner EW: Polyamine transport systems in mammalian cells and tissues. Methods Mol Biol. 2011, 720: 339-48. 10.1007/978-1-61779-034-8_21PubMed CentralView ArticlePubMedGoogle Scholar
- Uemura T, Yerushalmi HF, Tsaprailis G, Stringer DE, Pastorian KE, Hawel L, Byus CV, Gerner EW: Identification and characterization of a diamine exporter in colon epithelial cells. J Biol Chem. 2008, 283: 26428-35. 10.1074/jbc.M804714200PubMed CentralView ArticlePubMedGoogle Scholar
- Wallner S, Schmitz G: Plasmalogens the neglected regulatory and scavenging lipid species. Chem Phys Lipids. 2011, 164: 573-89. 10.1016/j.chemphyslip.2011.06.008View ArticlePubMedGoogle Scholar
- Pauwels EK, Kairemo K: Fatty acid facts, part II: role in the prevention of carcinogenesis, or, more fish on the dish?. Drug News Perspect. 2008, 21: 504-10. 10.1358/dnp.2008.21.9.1290819View ArticlePubMedGoogle Scholar
- Giros A, Grzybowski M, Sohn VR, Pons E, Fernandez-Morales J, Xicola RM, Sethi P, Grzybowski J, Goel A, Boland CR, Gassull MA, Llor X: Regulation of colorectal cancer cell apoptosis by the n-3 polyunsaturated fatty acids Docosahexaenoic and Eicosapentaenoic. Cancer Prev Res (Phila). 2009, 2: 732-42. 10.1158/1940-6207.CAPR-08-0197.View ArticleGoogle Scholar
- Smith RE, Lespi P, Di Luca M, Bustos C, Marra FA, de Alaniz MJ, Marra CA: A reliable biomarker derived from plasmalogens to evaluate malignancy and metastatic capacity of human cancers. Lipids. 2008, 43: 79-89. 10.1007/s11745-007-3133-6View ArticlePubMedGoogle Scholar
- Kuo YB, Chan CC, Chang CA, Fan CW, Hung RP, Hung YS, Chen KT, Yu JS, Chang YS, Chan EC: Identification of phospholipid scramblase 1 as a biomarker and determination of its prognostic value for colorectal cancer. Mol Med. 2011, 17: 41-7.PubMed CentralView ArticlePubMedGoogle Scholar
- Munn NJ, Arnio E, Liu D, Zoeller RA, Liscum L: Deficiency in ethanolamine plasmalogen leads to altered cholesterol transport. J Lipid Res. 2003, 44: 182-92. 10.1194/jlr.M200363-JLR200View ArticlePubMedGoogle Scholar
- Wood PL, Khan A, Mankidy R, Smith T, Goodenowe DB: Plasmalogen Deficit: A New and Testable Hypothesis for the Etiology of Alzheimer's Disease. Open Access in Alzheimers Disease-Book 2. 2011, ISBN 979-953-307-022-2,Google Scholar
- Wood PL, Smith T, Pelzer L, Goodenowe DB: Targeted metabolomic analyses of cellular models of pelizaeus-merzbacher disease reveal plasmalogen and myo-inositol solute carrier dysfunction. Lipids Health Dis. 2011, 10: 102- 10.1186/1476-511X-10-102PubMed CentralView ArticlePubMedGoogle Scholar
- Wood PL, Khan MA, Smith T, Ehrmantraut G, Jin W, Cui W, Braverman NE, Goodenowe DB: In vitro and in vivo plasmalogen replacement evaluations in rhizomelic chrondrodysplasia punctata and Pelizaeus-Merzbacher disease using PPI-1011, an ether lipid plasmalogen precursor. Lipids Health Dis. 2011, 10: 182-PubMed CentralView ArticlePubMedGoogle Scholar
- Xie X, Gillies RJ, Gerner EW: Characterization of a diamine exporter in Chinese hamster ovary cells and identification of specific polyamine substrates. J Biol Chem. 1997, 272: 20484-9. 10.1074/jbc.272.33.20484View ArticlePubMedGoogle Scholar
- Thompson P, Gerner EW: Of timing and surrogates - A way forward for cancer chemoprevention. Clin Cancer Res. 2011, 17: 3509-11. 10.1158/1078-0432.CCR-11-0643PubMed CentralView ArticlePubMedGoogle Scholar
- Gallesio C, Colombatto S, Modica R: Free and acetylated polyamines as markers of oral cavity tumors. Oral Surg Oral Med Oral Pathol. 1994, 77: 167-71. 10.1016/0030-4220(94)90280-1View ArticlePubMedGoogle Scholar
- Löser C, Fölsch UR, Paprotny C, Creutzfeldt W: Polyamine concentrations in pancreatic tissue, serum, and urine of patients with pancreatic cancer. Pancreas. 1990, 5: 119-27. 10.1097/00006676-199003000-00001View ArticlePubMedGoogle Scholar
- Löser C, Fölsch UR, Paprotny C, Creutzfeldt W: Polyamines in colorectal cancer. Evaluation of polyamine concentrations in the colon tissue, serum, and urine of 50 patients with colorectal cancer. Cancer. 1990, 65: 958-66. 10.1002/1097-0142(19900215)65:4<958::AID-CNCR2820650423>3.0.CO;2-ZView ArticlePubMedGoogle Scholar
- Chanda R, Ganguly AK: Diamine-oxidase activity and tissue di- and poly-amine contents of human ovarian, cervical and endometrial carcinoma. Cancer Lett. 1995, 89: 23-8.View ArticlePubMedGoogle Scholar
- Tjandrawinata RR, Hawel L, Byus CV: Regulation of putrescine export in lipopolysaccharide or IFN-gamma-activated murine monocytic-leukemic RAW 264 cells. J Immunol. 1994, 152: 3039-52.PubMedGoogle Scholar
- Hawel L, Tjandrawinata RR, Fukumoto GH, Byus CV: Biosynthesis and selective export of 1, 5-diaminopentane (cadaverine) in mycoplasma-free cultured mammalian cells. J Biol Chem. 1994, 269: 7412-8.PubMedGoogle Scholar
- Babbar N, Gerner EW, Casero RA: Induction of spermidine/spermine N1-acetyltransferase (SSAT) by aspirin in Caco-2 colon cancer cells. Biochem J. 2006, 394: 317-24. 10.1042/BJ20051298PubMed CentralView ArticlePubMedGoogle Scholar
- Grösch S, Tegeder I, Niederberger E, Bräutigam L, Geisslinger G: COX-2 independent induction of cell cycle arrest and apoptosis in colon cancer cells by the selective COX-2 inhibitor celecoxib. FASEB J. 2001, 15: 2742-4.PubMedGoogle Scholar
- Mankidy R, Ahiahonu PW, Ma H, Jayasinghe D, Ritchie SA, Khan MA, Su-Myat KK, Wood PL, Goodenowe DB: Membrane plasmalogen composition and cellular cholesterol regulation: a structure activity study. Lipids Health Dis. 2010, 9: 62- 10.1186/1476-511X-9-62PubMed CentralView ArticlePubMedGoogle Scholar
- Périchon R, Moser AB, Wallace WC, Cunningham SC, Roth GS, Moser HW: Peroxisomal disease cell lines with cellular plasmalogen deficiency have impaired muscarinic cholinergic signal transduction activity and amyloid precursor protein secretion. Biochem Biophys Res Commun. 1998, 248: 57-61. 10.1006/bbrc.1998.8909View ArticlePubMedGoogle Scholar
- Styger R, Wiesmann UN, Honegger UE: Plasmalogen content and beta-adrenoceptor signalling in fibroblasts from patients with Zellweger syndrome. Effects of hexadecylglycerol. Biochim Biophys Acta. 2002, 1585: 39-43. 10.1016/S1388-1981(02)00320-7View ArticlePubMedGoogle Scholar
- Vartanian T, Sprinkle TJ, Dawson G, Szuchet S: Oligodendrocyte substratum adhesion modulates expression of adenylate cyclase-linked receptors. Proc Natl Acad Sci USA. 1988, 85: 939-43. 10.1073/pnas.85.3.939PubMed CentralView ArticlePubMedGoogle Scholar
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