Polyunsaturated fatty acids (PUFAs) inhibit proliferation and induce differentiation in leukemia cells. To investigate the molecular mechanisms whereby fatty acids affect these processes, U937 leukemia cells were conditioned with stearic, oleic, linolenic, ␣-linolenic, arachidonic, eicosapentaenoic, and docosahexaenoic acids. PUFAs affected proliferation; eicosapentaenoic acid (EPA) was the most potent on cell cycle progression. EPA enhanced the expression of the myeloid lineage-specific transcription factors CCAAT/enhancer-binding proteins (C/EBP␤ and C/EBP␦), PU.1, and c-Jun, resulting in increased expression of the monocyte lineage-specific target gene, the macrophage colony-stimulating factor receptor. Indeed, it is known that PU.1 and C/EBPs interact with their consensus sequences on a small DNA fragment of macrophage colony-stimulating factor receptor promoter, which is a determinant for expression. We demonstrated that C/EBP␤ and C/EBP␦ bind the same response element as a heterodimer. We focused on the enhanced expression of C/EBP␦, which has been reported to be a tumor suppressor gene silenced by promoter hypermethylation in U937 cells. After U937 conditioning with EPA and bisulfite sequencing of the ؊370/؊20 CpG island on the C/EBP␦ promoter region, we found a site-specific CpG demethylation that was a determinant for the binding activity of Sp1, an essential factor for C/EBP␦ gene basal expression. Our results provide evidence for a new role of PUFAs in the regulation of gene expression. Moreover, we demonstrated for the first time that re-expression of the tumor suppressor C/EBP␦ is controlled by the methylation state of a site-specific CpG dinucleotide.

Although the mechanisms underlying FAS overexpression in cancer are largely unknown, recent evidence (7-10) strongly suggests that, as in liver, FAS gene transcription in tumor cells is modulated by the sterol regulatory element binding protein-1 (SREBP-1).SREBP-1 is synthesized as an integral protein of the endoplasmic reticulum membranes. As its C-terminal regulatory domain interacts with SREBP cleavage-activating protein (SCAP), the SREBP/SCAP complex migrates to the Golgi membranes, where a two-step cleavage catalyzed by a serine protease [site-1 protease (S1P)] and a metalloprotease [site-2 protease (S2P)] is responsible for the release of the N-terminal sequence of SREBP-1 in the cytoplasm. At the nuclear level, mature SREBP-1, a transcription factor of the basic helix-loop-helix leucine zipper family, activates genes encoding FAS and other lipogenic enzymes by interacting with sterol response elements present in their promoter region (11). Insulin and dietary carbohydrates activate FAS gene transcription by this mechanism, whereas dietary polyunsaturated fatty acids (PUFAs) of both -6 and -3 series negatively modulate FAS expression by depressing SREBP-1 mRNA level and reducing the cleavage of the native protein (12, 13).Recently, it was found that pharmacological inhibitors of FAS are selectively cytotoxic to tumor cells in culture and in vivo (14-17). In particular, the FAS inhibitors cerulenin (2 S ,3 R -epoxy-4-oxo-7 E ,10 E -dodecadienamide) and C75 (3-carboxy-4-octyl-2-methylenebutyrolactone) induce Abbreviations: ACC, acetyl-CoA carboxylase; C/EBP, CCAAT enhancer binding protein; COX-2, cyclooxygenase-2; CPT-1, carnitine palmitoyltransferase-1; FAS, fatty acid synthetase; PPAR ␣ , peroxisome proliferator-activated receptor-␣ ; SCAP, sterol regulatory element binding protein-1 cleavage activating protein; S1P, site-1 protease; S2P, site-2 protease; SREBP-1, sterol regulatory element binding protein-1.

No abstract

Epigenetic alterations, including aberrant DNA methylation, contribute to tumor development and progression. Silencing of tumor suppressor genes may be ascribed to promoter DNA hypermethylation, a reversible phenomenon intensely investigated as potential therapeutic target. Previously, we demonstrated that eicosapentaenoic acid (EPA) exhibits a DNA demethylating action that promotes the re-expression of the tumor suppressor gene CCAAT/enhancer-binding protein δ (C/EBPδ). The C/EBPβ/C/EBPδ heterodimer formed appears essential for the monocyte differentiation commitment. The present study aims to evaluate the effect of EPA on RAS/extracellular signal regulated kinases (ERK1/2)/C/EBPβ pathway, known to be induced during the monocyte differentiation program. We found that EPA conditioning of U937 leukemia cells activated RAS/ERK/C/EBPβ pathway, increasing the C/EBPβ and ERK1/2 active phosphorylated forms. Transcriptional induction of the upstream activator H-Ras gene resulted in increased expression of H-Ras protein in the active pool of non raft membrane fraction. H-Ras gene analysis identified an hypermethylated CpG island in intron 1 that can affect the DNA-protein interaction modifying RNA polymerase II (RNAPII) activity. EPA treatment demethylated almost completely this CpG island, which was associated with an enrichment of active RNAPII. The increased binding of the H-Ras transcriptional regulator p53 to its consensus sequence within the intronic CpG island further confirmed the effect of EPA as demethylating agent. Our results provide the first evidence that an endogenous polyunsaturated fatty acid (PUFA) promotes a DNA demethylation process responsible for the activation of RAS/ERK/C/EBPβ pathway during the monocyte differentiation commitment. The new role of EPA as demethylating agent paves the way for studying PUFA action when aberrant DNA methylation is involved.

In cancer cells, global genomic hypomethylation is found together with localized hypermethylation of CpG islands within the promoters and regulatory regions of silenced tumor suppressor genes. Demethylating agents may reverse hypermethylation, thus promoting gene re-expression. Unfortunately, demethylating strategies are not efficient in solid tumor cells. DNA demethylation is mediated by ten-eleven translocation enzymes (TETs). They sequentially convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), which is associated with active transcription; 5-formylcytosine; and finally, 5-carboxylcytosine. Although α-linolenic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid, the major n-3 polyunsaturated fatty acids, have anti-cancer effects, their action, as DNA-demethylating agents, has never been investigated in solid tumor cells. Here, we report that EPA demethylates DNA in hepatocarcinoma cells. EPA rapidly increases 5hmC on DNA, inducing p21 gene expression, which slows cancer cell-cycle progression. We show that the underlying molecular mechanism involves TET1. EPA simultaneously binds peroxisome proliferator-activated receptor γ (PPARγ) and retinoid X receptor α (RXRα), thus promoting their heterodimer and inducing a PPARγ-TET1 interaction. They generate a TET1-PPARγ-RXRα protein complex, which binds to a hypermethylated CpG island on the p21 gene, where TET1 converts 5mC to 5hmC. In an apparent shuttling motion, PPARγ and RXRα leave the DNA, whereas TET1 associates stably. Overall, EPA directly regulates DNA methylation levels, permitting TET1 to exert its anti-tumoral function.-Ceccarelli, V., Valentini, V., Ronchetti, S., Cannarile, L., Billi, M., Riccardi, C., Ottini, L., Talesa, V. N., Grignani, F., Vecchini, A., Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism.