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• W2091217195 abstract "Malignant gliomas (MG) are highly infiltrative tumors that consistently recur despite aggressive treatment. Brain fatty acid-binding protein (FABP7), which binds docosahexaenoic acid (DHA) and arachidonic acid (AA), localizes to sites of tumor infiltration and is associated with a poor prognosis in MG. Manipulation of FABP7 expression in MG cell lines affects cell migration, suggesting a role for FABP7 in tumor infiltration and recurrence. Here, we show that DHA inhibits and AA stimulates migration in an FABP7-dependent manner in U87 MG cells. We demonstrate that DHA binds to and sequesters FABP7 to the nucleus, resulting in decreased cell migration. This anti-migratory effect is partially dependent on peroxisome proliferator-activated receptor γ, a DHA-activated transcription factor. Conversely, AA-bound FABP7 stimulates cell migration by activating cyclooxygenase-2 and reducing peroxisome proliferator-activated receptor γ levels. Our data provide mechanistic insight as to why FABP7 is associated with a poor prognosis in MG and suggest that relative levels of DHA and AA in the tumor environment can make a profound impact on tumor growth properties. We propose that FABP7 and its fatty acid ligands may be key therapeutic targets for controlling the dissemination of MG cells within the brain. Malignant gliomas (MG) are highly infiltrative tumors that consistently recur despite aggressive treatment. Brain fatty acid-binding protein (FABP7), which binds docosahexaenoic acid (DHA) and arachidonic acid (AA), localizes to sites of tumor infiltration and is associated with a poor prognosis in MG. Manipulation of FABP7 expression in MG cell lines affects cell migration, suggesting a role for FABP7 in tumor infiltration and recurrence. Here, we show that DHA inhibits and AA stimulates migration in an FABP7-dependent manner in U87 MG cells. We demonstrate that DHA binds to and sequesters FABP7 to the nucleus, resulting in decreased cell migration. This anti-migratory effect is partially dependent on peroxisome proliferator-activated receptor γ, a DHA-activated transcription factor. Conversely, AA-bound FABP7 stimulates cell migration by activating cyclooxygenase-2 and reducing peroxisome proliferator-activated receptor γ levels. Our data provide mechanistic insight as to why FABP7 is associated with a poor prognosis in MG and suggest that relative levels of DHA and AA in the tumor environment can make a profound impact on tumor growth properties. We propose that FABP7 and its fatty acid ligands may be key therapeutic targets for controlling the dissemination of MG cells within the brain. Anaplastic astrocytoma (grade III astrocytoma) and glioblastoma multiforme (grade IV astrocytoma), collectively called malignant gliomas (MG), 2The abbreviations used are: MGmalignant gliomaFABPfatty acid-binding proteinDHAdocosahexaenoic acidAAarachidonic acidPPARperoxisome proliferator-activated receptorPUFApolyunsaturated fatty acidPApalmitic acidPGprostaglandinNLSnuclear localization signal. are the most common cancers of the central nervous system (CNS). The prognosis for these cancers is dismal, with median survival times of 30 months for grade III astrocytoma and less than 1 year for grade IV astrocytoma (1Lin C.L. Lieu A.S. Lee K.S. Yang Y.H. Kuo T.H. Hung M.H. Loh J.K. Yen C.P. Chang C.Z. Howng S.L. Hwang S.L. Surg. Neurol. 2003; 60: 402-406Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). Despite aggressive treatment involving surgical resection, radiation therapy, and adjuvant chemotherapy using nitrosourea-based compounds and temozolamide (2Mason W.P. Maestro R.D. Eisenstat D. Forsyth P. Fulton D. Laperrière N. Macdonald D. Perry J. Thiessen B. Curr. Oncol. 2007; 14: 110-117Crossref PubMed Scopus (92) Google Scholar), these highly infiltrative tumors consistently recur in the brain at sites that can be either proximal or distal to the original tumor mass. Thus, improving patient survival will likely depend on developing new therapies that target infiltrative cells while minimizing damage to normal brain tissue. malignant glioma fatty acid-binding protein docosahexaenoic acid arachidonic acid peroxisome proliferator-activated receptor polyunsaturated fatty acid palmitic acid prostaglandin nuclear localization signal. Brain fatty acid-binding protein (FABP7; BLBP) is expressed in astrocytoma tumor biopsies as well as in a subset of MG cell lines (3Godbout R. Bisgrove D.A. Shkolny D. Day 3rd, R.S. Oncogene. 1998; 16: 1955-1962Crossref PubMed Scopus (73) Google Scholar), and FABP7 is up-regulated in brain tumor tissue compared with normal adult brain (4Liang Y. Diehn M. Watson N. Bollen A.W. Aldape K.D. Nicholas M.K. Lamborn K.R. Berger M.S. Botstein D. Brown P.O. Israel M.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5814-5819Crossref PubMed Scopus (403) Google Scholar, 5Tso C.L. Shintaku P. Chen J. Liu Q. Liu J. Chen Z. Yoshimoto K. Mischel P.S. Cloughesy T.F. Liau L.M. Nelson S.F. Mol. Cancer Res. 2006; 4: 607-619Crossref PubMed Scopus (188) Google Scholar). FABP7 expression has been associated with decreased survival times and/or tumor progression in patients with grade IV astrocytoma (4Liang Y. Diehn M. Watson N. Bollen A.W. Aldape K.D. Nicholas M.K. Lamborn K.R. Berger M.S. Botstein D. Brown P.O. Israel M.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5814-5819Crossref PubMed Scopus (403) Google Scholar, 6Kaloshi G. Mokhtari K. Carpentier C. Taillibert S. Lejeune J. Marie Y. Delattre J.Y. Godbout R. Sanson M. J. Neurooncol. 2007; 84: 245-248Crossref PubMed Scopus (69) Google Scholar), melanoma, basal-type breast cancer, and renal cell carcinoma (7Zhang H. Rakha E.A. Ball G.R. Spiteri I. Aleskandarany M. Paish E.C. Powe D.G. Macmillan R.D. Caldas C. Ellis I.O. Green A.R. Breast Cancer Res. Treat. 2010; 121: 41-51Crossref PubMed Scopus (47) Google Scholar, 8Seliger B. Lichtenfels R. Atkins D. Bukur J. Halder T. Kersten M. Harder A. Ackermann A. Malenica B. Brenner W. Zobawa M. Lottspeich F. Proteomics. 2005; 5: 2631-2640Crossref PubMed Scopus (33) Google Scholar, 9Slipicevic A. Jørgensen K. Skrede M. Rosnes A.K. Trøen G. Davidson B. Flørenes V.A. BMC Cancer. 2008; 8: 276Crossref PubMed Scopus (49) Google Scholar). Introduction of FABP7 into FABP7-negative MG cells confers a pro-migratory/pro-invasive phenotype to the cells, whereas knockdown of FABP7 in MG cells that naturally express FABP7 results in reduced migration/invasion (4Liang Y. Diehn M. Watson N. Bollen A.W. Aldape K.D. Nicholas M.K. Lamborn K.R. Berger M.S. Botstein D. Brown P.O. Israel M.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5814-5819Crossref PubMed Scopus (403) Google Scholar, 10Mita R. Coles J.E. Glubrecht D.D. Sung R. Sun X. Godbout R. Neoplasia. 2007; 9: 734-744Crossref PubMed Scopus (64) Google Scholar). These combined data suggest that FABP7 may enhance the infiltrative and/or invasive properties of tumor cells, thereby contributing to tumor recurrence and decreased patient survival. FABP7 is a member of the FABP family of lipid chaperones involved in the uptake and intracellular trafficking of fatty acids. In vitro ligand binding studies have shown that the polyunsaturated fatty acid (PUFA) ω-3-docosahexaenoic acid (DHA; 22:6) is the preferred ligand of FABP7. FABP7 also binds ω-6-arachidonic acid (AA; 20:4), albeit with an ∼4-fold lower affinity (11Balendiran G.K. Schnutgen F. Scapin G. Borchers T. Xhong N. Lim K. Godbout R. Spener F. Sacchettini J.C. J. Biol. Chem. 2000; 275: 27045-27054Abstract Full Text Full Text PDF PubMed Google Scholar). DHA and AA have been shown to have opposite effects on tumor growth, with DHA inhibiting growth and AA promoting growth (12Larsson S.C. Kumlin M. Ingelman-Sundberg M. Wolk A. Am. J. Clin. Nutr. 2004; 79: 935-945Crossref PubMed Scopus (775) Google Scholar). FABPs, found in both the nucleus and cytoplasm, are believed to play a role in gene regulation by activating peroxisome proliferator-activated receptors (PPARs), nuclear receptors that function as transcription factors. Specifically, liver FABP (FABP1) has been shown to bind and activate PPARα and PPARγ (13Wolfrum C. Borrmann C.M. Borchers T. Spener F. Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 2323-2328Crossref PubMed Scopus (419) Google Scholar). Adipocyte FABP (FABP4) and keratinocyte FABP (FABP5) bind PPARγ and PPARβ, respectively (14Tan N.S. Shaw N.S. Vinckenbosch N. Liu P. Yasmin R. Desvergne B. Wahli W. Noy N. Mol. Cell. Biol. 2002; 22: 5114-5127Crossref PubMed Scopus (400) Google Scholar, 15Helledie T. Jørgensen C. Antonius M. Krogsdam A.M. Kratchmarova I. Kristiansen K. Mandrup S. Mol. Cell. Biochem. 2002; 239: 157-164Crossref PubMed Scopus (26) Google Scholar), whereas FABP7 interacts with PPARγ (16Adida A. Spener F. Biochim. Biophys. Acta. 2006; 1761: 172-181Crossref PubMed Scopus (70) Google Scholar). It has been postulated that nuclear FABPs can deliver their fatty acid ligands to PPARs, thereby regulating PPAR transcriptional activity. Increased expression of PPARα is associated with a worse prognosis in MG (17Benedetti E. Galzio R. Laurenti G. D'Angelo B. Melchiorre E. Cifone M.G. Fanelli F. Muzi P. Coletti G. Alecci M. Sotgiu A. Cerù M.P. Cimini A. Int. J. Immunopathol. Pharmacol. 2010; 23: 235-246Crossref PubMed Scopus (23) Google Scholar), whereas PPARγ is generally associated with growth arrest and apoptosis in these tumors (18Papi A. Tatenhorst L. Terwel D. Hermes M. Kummer M.P. Orlandi M. Heneka M.T. J. Neurochem. 2009; 109: 1779-1790Crossref PubMed Scopus (54) Google Scholar, 19Chearwae W. Bright J.J. Br. J. Cancer. 2008; 99: 2044-2053Crossref PubMed Scopus (78) Google Scholar). Although relationships between specific fatty acids and FABPs, FABPs and cancer, and fatty acids and cancer have been described previously, there has been no concerted effort to investigate the interdependence of fatty acids and FABPs on tumorigenic properties. In a previous study, Wang et al. (20Wang M. Liu Y.E. Ni J. Aygun B. Goldberg I.D. Shi Y.E. Cancer Res. 2000; 60: 6482-6487PubMed Google Scholar) demonstrated ∼50% growth inhibition of breast cancer cells stably transfected with an FABP7 expression construct in the presence of DHA. Furthermore, treatment of FABP5-expressing PC12 cells with DHA increased neurite extension (21Liu J.W. Almaguel F.G. Bu L. De Leon D.D. De Leon M. J. Neurochem. 2008; 106: 2015-2029PubMed Google Scholar). These data highlight the importance of FABPs in determining tumor cell response to fatty acids. Here, we examine the effect of DHA and AA on FABP7-mediated cell migration in MG. We describe an inhibitory role for DHA in FABP7-mediated cell migration and a permissive role for AA in FABP7-mediated cell migration. Intriguingly, DHA-mediated inhibition of cell migration functions through PPARγ and requires nuclear localization of FABP7, whereas stimulation of cell migration by FABP7 and AA is dependent on activation of cyclooxygenase 2 (COX-2) and prostaglandin E2 (PGE2) production. We propose a model whereby relative levels of DHA and AA, and the fatty acid-dependent subcellular distribution of FABP7, determine the migratory potential of MG cells. The importance of our findings resonates 2-fold as they provide a molecular mechanism for FABP7-induced MG cell migration and point to the potential use of DHA as an anti-infiltrative therapeutic agent in the treatment of MG. The origin and FABP7 status of MG cell lines U87, U251, U373, and M049, as well as the generation of stable U87 clonal populations transfected with either empty pREP4 vector (FABP7(−)) or a pREP4-FABP7 expression construct (FABP7(+)), have been described previously (10Mita R. Coles J.E. Glubrecht D.D. Sung R. Sun X. Godbout R. Neoplasia. 2007; 9: 734-744Crossref PubMed Scopus (64) Google Scholar, 22Bisgrove D.A. Monckton E.A. Packer M. Godbout R. J. Biol. Chem. 2000; 275: 30668-30676Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 23Brun M. Coles J.E. Monckton E.A. Glubrecht D.D. Bisgrove D. Godbout R. J. Mol. Biol. 2009; 391: 282-300Crossref PubMed Scopus (51) Google Scholar). Transient transfection of U87 cells was by calcium phosphate-DNA precipitation, with 40–50% transfection efficiency. Transiently transfected cells were analyzed 48 h after removal of the DNA. Unless otherwise stated, cells were cultured in Dulbecco's modified essential medium (DMEM) supplemented with 10% FCS, 100 μg/ml streptomycin, and 100 units/ml penicillin. Whole cell lysates were prepared by incubating cells on ice for 20 min in 0.5 m Tris-HCl, pH 7.5, 0.15 m NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 1 mm NaF, 0.1% SDS, 1× protease inhibitor mixture (Roche Applied Sci), 1 mm PMSF, 2 mm DTT. Nuclear cell extracts were prepared by lysing cells at 4 °C in 10 mm HEPES, pH 7.9, 10 mm KCl, 0.1 mm EDTA, 0.4% Nonidet P-40, 1 mm DTT, 0.5 mm PMSF. Nuclei were collected by centrifugation (15,000 × g at 4 °C for 3 min) and lysed by vigorous shaking in 20 mm HEPES, pH 7.9, 0.4 m NaCl, 1 mm EDTA, 10% glycerol, 1 mm DTT, 0.5 mm PMSF. Nuclear lysates were centrifuged at 14,000 × g for 5 min at 4 °C, and the supernatants were collected. Lysates were electrophoresed in a 13.5% SDS-polyacrylamide gel followed by electroblotting to nitrocellulose membranes. Membranes were immunostained with rabbit anti-FABP7 (3Godbout R. Bisgrove D.A. Shkolny D. Day 3rd, R.S. Oncogene. 1998; 16: 1955-1962Crossref PubMed Scopus (73) Google Scholar), mouse anti-HA (Santa Cruz Biotechnology), mouse anti-actin (Sigma-Aldrich), rabbit anti-FABP5 (HyCult Biotechnology), goat anti-COX-2 (sc-1747; Santa Cruz Biotechnology), mouse anti-PPARα (Chemicon), mouse anti-PPARβ (Chemicon), or mouse anti-PPARγ (Chemicon) antibodies. Primary antibodies were detected with horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch) using the ECL detection system (GE Healthcare). Twenty thousand cells were plated in DMEM (without FCS) in the top chamber of HTS multiwell inserts with a porous 8 μm PET membrane separating the top and bottom chambers (Transwell; BD Biosciences). Cells were incubated for 6 h in a CO2 incubator to allow cell migration to the bottom chamber containing chemoattractant (DMEM plus 10% FCS). Cells that migrated to the bottom chamber were fixed and stained with 2% crystal violet in 20% methanol. The cells were photographed using a 2.5× lens and four frames combined to reconstruct the entire well. Cells were then counted using Metamorph version 7.7 imaging software. Cells were fixed in 1% paraformaldehyde for 10 min and then permeabilized in PBS + 0.5% Triton X-100 for 5 min. Affinity-purified rabbit anti-FABP7 antibody was used at a 1:200 dilution, followed by secondary Alexa 488-conjugated goat anti-rabbit secondary antibody (Cedarlane Laboratories). All images were collected on a Zeiss LSM 510 confocal microscope with a 40×/1.3 oil immersion lens. Lipidex® 1000 (Sigma) was used to delipidate recombinant FABP7 and carry out the fatty acid binding assays. Lipidex 1000 removes unbound and/or protein-bound fatty acids from aqueous solutions in a temperature-dependent manner. For delipidation, recombinant GST-tagged FABP7 was incubated in 1.0-ml amber glass vials along with 50% w/v Lipidex in 10 mm potassium phosphate buffer, pH 7.4, for 10 min at 37 °C. The Lipidex/protein mixture was then spun down for 2 min at 4,000 × g, and the incubation repeated twice using fresh Lipidex-conjugated beads. For fatty acid binding assays, delipidated FABP7 was incubated in 10 mm potassium phosphate buffer, pH 7.4, with varying concentrations of [1-14C]DHA (Moravek Biochemicals) and incubated at 37 °C for 10 min. Unbound fatty acids were removed from the solution by adding 50 μl of an ice-cold Lipidex/buffer suspension (1:1, w/v) followed by a 10-min incubation at 4 °C. The mixture was then centrifuged for 5 min at 4,000 × g, and fatty acid binding was calculated from the amount of radioactivity present in the supernatant as measured by a scintillation counter. Graphs were generated and analyzed using GraphPad Prism software version 5. Cells were transfected with 10 nm of the following Stealth siRNAs (Invitrogen) using LipofectamineTM RNAiMAX (Invitrogen): FABP7 (5′-CAAACCAACGGUAAUUAUCAGUCAA-3′ (NM_001446_stealth_405)); PPARγ (5′-CCAGUGGUUGCAGAUUACAAGUAUG-3′ (NM_13872_stealth_425)); PPARγ (5′-AGGGAGUUUCUAAAGAGCCUGCGAA-3′ (NM_13772_stealth_1295)); PPARβ (5′-CCACUACGGUGUUCAUGCAUGUGAG-3′ (NM_006238_stealth_561)); PPARβ (5′-UCAGUGAUAUCAUUGAGCCUAAGUU-3′ (NM_006238_stealth_1280)); scrambled siRNA (StealthTM RNAi Negative Control Low and Medium GC Duplex). The Transwell migration assay was carried out 72 h post-transfection using 20,000 cells/well. The remaining cells were used for the preparation of cell lysates and used for Western blot analysis to verify knockdown. PGE2 levels were determined by ELISA using the protocol supplied by the manufacturer (prostaglandin E2 Biotrak Enzyme Immunoassay System, GE Healthcare). Cells were seeded in triplicate on 96-well plates at 50,000 cells/well and cultured for 24 h. Plates were read at 450 nm using a microplate reader (FLUOstar OPTIMA, BMG LABTECH). Fatty acids (Sigma) were dissolved in ethanol and then complexed to fatty acid-free BSA (Roche Applied Science) over a steady stream of nitrogen gas. The final concentration of ethanol in our experiments was 0.1%. NS398 (Sigma) was dissolved in DMSO (Sigma) to generate a 200 mm (1000×) stock solution. The final concentration of DMSO in the medium was 0.1%. The 400-bp human FABP7 cDNA encoding the entire open reading frame was inserted into the pcDNA3.1 vector at the EcoRV site. To generate the nuclear localization signal (NLS) mutant, site-directed mutagenesis of FABP7 at K21A (AAG→GCG), R30A (AGG→GCG), and Q31A (CAG→GCG) was carried out by sequential PCR (24Cormack B. Castaño I. Methods Enzymol. 2002; 350: 199-218Crossref PubMed Scopus (21) Google Scholar) using partially complementary primers carrying the appropriate point mutations. These primers were used in conjunction with pcDNA3.1 vector primers to generate DNA fragments corresponding to full-length FABP7. To generate the nonfatty acid-binding mutant, the same technique was applied, using primers carrying the following point mutations: F104A (TTT→AGA), R126A (CGC→GCC), and Y128A (TAT→GCT). These primers, which contained an XhoI recognition site, were used along with primers to the pcDNA3.1 vector sequence spanning the BamHI site to allow directional cloning into pcDNA3.1. The FABP7 cDNA inserts were sequenced to ensure that they were error-free. Expression of mutant FABP7 was confirmed by transfection into U87MG and Western blot analysis. Previous work has shown that stable transfection of an FABP7 expression construct into FABP7(−) U87MG cells results in increased motility and migration (10Mita R. Coles J.E. Glubrecht D.D. Sung R. Sun X. Godbout R. Neoplasia. 2007; 9: 734-744Crossref PubMed Scopus (64) Google Scholar). In vitro ligand binding studies indicate that FABP7 binds both DHA and AA, with equilibrium dissociation constants (Kd) of 53.4 and 207 nm, respectively (11Balendiran G.K. Schnutgen F. Scapin G. Borchers T. Xhong N. Lim K. Godbout R. Spener F. Sacchettini J.C. J. Biol. Chem. 2000; 275: 27045-27054Abstract Full Text Full Text PDF PubMed Google Scholar). We therefore used the Transwell assay to examine the effect of DHA and AA on the migration of FABP7(−) and FABP7(+) U87 cells. Cells were cultured in growth medium (DMEM plus 10% FCS) supplemented with 60 μm BSA-complexed DHA, AA, palmitic acid (PA; 16:0), or BSA alone for 24 h. As demonstrated in Fig. 1A, none of the fatty acids tested had an effect on FABP7(−) U87 cell migration, with an average of ∼250 cells migrating through the upper chamber of Transwell inserts. As expected, FABP7-expressing U87 cells cultured in 10% FCS supplemented with BSA showed increased migration compared with FABP7(−) U87 cells (1553 versus 294 cells/well). Growth in PA had no effect on FABP7(+) U87 cell migration; however, growth in DHA resulted in a 5-fold decrease in cell migration, with an average of 279 cells migrating to the bottom chamber compared with 1553 cells in the BSA control (p < 0.01) (Fig. 1A). In contrast, FABP7(+) U87 cells cultured in AA showed a small but significant increase in cell migration, with 1812 cells/well migrating to the bottom chamber compared with 1553 in the BSA control (p < 0.01). These results indicate a strong link between FABP7-mediated cell migration and the presence of DHA or AA. Studying clonal populations of U87 cells that differ in whether they have been transfected with empty vector or an FABP7 expression construct represents a highly controlled experimental situation. To broaden the scope of our study, we examined the effect of AA and DHA on three MG cell lines that naturally express FABP7: U251, U373, and M049 (Fig. 2A). U251 and U373 cultured in the presence of BSA had migratory activities that were similar to that of FABP7(+) U87 cells (Fig. 2A). Although growth of U251 and U373 cells in AA did not significantly increase cell migration, U251 and U373 cells cultured in DHA showed the same decrease in migration observed for FABP7(+) U87 cells. In contrast, M049 cells showed significantly reduced migration compared with the other FABP7-expressing MG cell lines. Similar to FABP7(−) U87 cells, neither DHA nor AA had an effect on M049 cell migration. These results indicate that factors other than FABP7 can affect MG cell migration. Of note, FABP5 is highly expressed in M049 compared with U251 and U373 (Fig. 2A). We then used siRNA to reduce FABP7 levels in U251 cells. Knockdown of FABP7 resulted in a significant decrease in cell migration, with 641 FABP7(↓) U251 cells migrating to the bottom chamber in comparison with 1490 cells for U251 control (p < 0.01) (Fig. 2B). Growth in the presence of DHA or AA had little effect on the migration of FABP7(↓) U251 cells, in keeping with the results obtained with FABP7(−) U87 cells. Because there was general, if not complete, agreement between the results obtained with U87, U251, and U373, we pursued our analyses with the U87-FABP7(−) and U87-FABP7(+) clonal populations. Fetal calf serum contains >20 different fatty acids, including DHA and AA at concentrations of 4 and 11.5 μm, respectively. To study the effects of DHA and AA on migration in the absence of FCS, U87-FABP7(−) and U87-FABP7(+) MG cells were cultured in DMEM without FCS for 24 h and then treated with DHA, AA, PA, or BSA in the absence of FCS for 24 h (i.e. 48-h total serum starvation). Although there was a 30% decrease in the number of serum-starved BSA-treated U87-FABP7(+) cells that migrated to the bottom chamber compared with cells cultured in 10% FCS (Fig. 1B), the overall trend was similar to that observed for cells grown in the presence of FCS, with DHA inhibiting and AA stimulating cell migration. There was an average of 1202 cells/well for the BSA control compared with 199 cells/well for DHA-treated cells and 1446 cells/well for AA-treated cells. Again, PA treatment did not affect cell migration, and none of the fatty acids affected the migration of U87-FABP7(−) cells. These results indicate that DHA and AA can regulate FABP7-mediated U87 cell migration. A number of studies have shown that ω-3 fatty acids have anti-tumorigenic properties, inhibiting tumor growth and preventing vascularization and cell migration. In contrast, ω-6 fatty acids have pro-tumorigenic properties. It has been postulated that the ratio of ω-3 to ω-6 fatty acids is key to the pathogenesis of many diseases, including cancer (25Simopoulos A.P. World Rev. Nutr. Diet. 2009; 99: 1-16Crossref PubMed Scopus (105) Google Scholar). To address whether the ratio of DHA:AA, as opposed to individual concentrations of DHA or AA, is driving the effects observed in Fig. 1, A and B, we treated U87-FABP7(+) MG cells with varying concentrations of both DHA and AA, and we measured their migration using the Transwell assay (Fig. 1C). The migration of U87-FABP7(+) cells cultured in growth medium supplemented with 60 μm each of DHA and AA was inhibited by 75%, with an average of 371 cells/well migrating to the bottom chamber compared with 1513 cells/well in the untreated control (Fig. 1C). Cell migration was also inhibited by 74% when the concentration of DHA was halved (30 μm DHA; 60 μm AA). However, when levels of AA exceeded those of DHA by 3-fold or more, the inhibitory effects of DHA on cell migration were negated, with 1596 cells/well migrating to the bottom chamber when the DHA:AA ratio was 1:3 (30 μm DHA; 90 μm AA), and 1646 cells/well migrating to the bottom chamber with a DHA:AA ratio of 1:4 (30 μm DHA; 120 μm AA). These results are expected in light of in vitro data indicating that DHA has a four times greater binding affinity for FABP7 than AA (11Balendiran G.K. Schnutgen F. Scapin G. Borchers T. Xhong N. Lim K. Godbout R. Spener F. Sacchettini J.C. J. Biol. Chem. 2000; 275: 27045-27054Abstract Full Text Full Text PDF PubMed Google Scholar). Increasing the concentration of DHA, from 60 to 120 μm, in the presence of 30 μm AA, did not result in further inhibition of cell migration (Fig. 1C). PA at concentrations ranging from 30 to 120 μm had no effect on cell migration (Fig. 3). Based on cellular morphology and growth rate, high concentrations of fatty acids had no adverse effect on the survival of FABP7(+) U87 cells. These combined data suggest that different pathways are activated when FABP7 is bound to DHA or AA, with DHA inhibiting and AA stimulating cell migration. FABPs play an important role in the transport of their fatty acid ligands to various subcellular compartments, including endoplasmic reticulum, mitochondria, and nucleus. FABP7 is found in both the cytoplasm and nucleus of MG cells, with some tumors preferentially expressing FABP7 in either the nucleus or cytoplasm (4Liang Y. Diehn M. Watson N. Bollen A.W. Aldape K.D. Nicholas M.K. Lamborn K.R. Berger M.S. Botstein D. Brown P.O. Israel M.A. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 5814-5819Crossref PubMed Scopus (403) Google Scholar, 6Kaloshi G. Mokhtari K. Carpentier C. Taillibert S. Lejeune J. Marie Y. Delattre J.Y. Godbout R. Sanson M. J. Neurooncol. 2007; 84: 245-248Crossref PubMed Scopus (69) Google Scholar). To investigate whether the subcellular localization of FABP7 might be dependent on availability of specific fatty acid ligands, we examined the subcellular distribution of FABP7 in U87-FABP7(+) and U251, in the presence and absence of DHA and AA. When cells were cultured in medium depleted of fatty acids (i.e. serum-starved for 24 h), FABP7 was primarily found in the cytoplasm (Fig. 4, A and D). Addition of 60 μm DHA to DMEM resulted in FABP7 localization to the nucleus (Fig. 4, B and E), suggesting that DHA-bound FABP7 is sequestered to the nucleus. In contrast, a relatively uniform distribution of FABP7 was observed throughout the cell in the presence of 60 μm AA (Fig. 4, C and F). Alterations in the subcellular distribution of FABP7 were not accompanied by any changes in the levels of FABP7 protein (Fig. 5). The dynamic shuttling of FABP7 from the cytoplasm to the nucleus in the presence of DHA (and to a lesser extent AA) suggests a role for FABP7 in delivering its fatty acid ligands to the nucleus.FIGURE 5Western blot analysis of FABP7 in U87-FABP7(+) stable transfectants treated with 60 μm BSA, AA, DHA, or PA. Whole cell lysates (25 μg/lane) were prepared from U87-FABP7(+) and electrophoresed in a 13.5% SDS-polyacrylamide gel. Proteins were transferred to nitrocellulose membranes and sequentially immunostained with rabbit anti-FABP7 antibody and mouse anti-actin antibody, followed by HRP-conjugated secondary antibodies. C, cytoplasmic; N, nuclear.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Based on x-ray crystallography, Balendiran et al. (11Balendiran G.K. Schnutgen F. Scapin G. Borchers T. Xhong N. Lim K. Godbout R. Spener F. Sacchettini J.C. J. Biol. Chem. 2000; 275: 27045-27054Abstract Full Text Full Text PDF PubMed Google Scholar) predicted that the amino acid Phe-104 is essential for the formation of π-π interactions between FABP7 and DHA, whereas the guanidinium group of Arg-126 and the hydroxyl group of Tyr-128 were predicted to bind directly to the carboxylate moiety of DHA. We therefore mutated the FABP7 Phe-104/Arg-126/Tyr-128 residues to Ala-104/Ala-126/Ala-128 (FABP7FAB) to gain further insight into the importance of fatty acid binding to FABP7-mediated migration in MG cells. FABP7FAB was cloned into the pcDNA3.1 expression vector and transfected into U87-FABP7(−) MG cells. Western blot analysis demonstrated the presence of FABP7FAB in the cytoplasm with little mutant protein in the nucleus (Fig. 6). The abundance of FABP7FAB in the cytoplasm indicates that any structural changes resulting from the site-directed mutagenesis had little or no effect on protein stability. Subcellular localization experiments verified that FABP7FAB mostly resides in the cytoplasm, in contrast to wild-type FABP7 (FABP7WT), which is abundant in the nucleus (Fig. 7, A and B). These findings are in keeping with our previous results indicating that serum starvation prevents FABP7 from accumulating in the nucleus.FIGURE 7Fatty acid binding is required for migration. Subcellular localization of FABP7 in U87MG cells transiently transfected with pcDNA3.1-FABP7WT (A) or pcDNA3.1-FABP7FAB (B) expression constructs. Transfection efficiency was ∼40%. Immunofluorescence was carried out 48 h after transfection using anti-FABP7 antibody followed by Alexa 488-conjugated secondary antibody. DNA was counterstained with DAPI. C, migration of U87MG cells transfected with empty vector, pcDNA3.1-FABP7WT, and pcDNA3.1-FABP7FAB was measured using" @default.
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• W2091217195 date "2010-11-01" @default.
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• W2091217195 title "Brain Fatty Acid-binding Protein and ω-3/ω-6 Fatty Acids" @default.
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