The Polyunsaturated Fatty Acid EPA, but Not DHA, Enhances Neurotrophic Factor Expression through Epigenetic Mechanisms and Protects against Parkinsonian Neuronal Cell Death.

ω-3 Polyunsaturated fatty acids (PUFAs) have been found to exert many actions, including neuroprotective effects. In this regard, the exact molecular mechanisms are not well understood. Parkinson's disease (PD) is the second most common age-related neurodegenerative disease. Emerging evidence supports the hypothesis that PD is the result of complex interactions between genetic abnormalities, environmental toxins, mitochondrial dysfunction, and other cellular processes, such as DNA methylation. In this context, BDNF (brain-derived neurotrophic factor) and GDNF (glial cell line-derived neurotrophic factor) have a pivotal role because they are both involved in neuron differentiation, survival, and synaptogenesis. In this study, we aimed to elucidate the potential role of two PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and their effects on BDNF and GDNF expression in the SH-SY5Y cell line. Cell viability was determined using the MTT assay, and flow cytometry analysis was used to verify the level of apoptosis. Transmission electron microscopy was performed to observe the cell ultrastructure and mitochondria morphology. BDNF and GDNF protein levels and mRNA were assayed by Western blotting and RT-PCR, respectively. Finally, methylated and hydroxymethylated DNA immunoprecipitation were performed in the BDNF and GDNF promoter regions. EPA, but not DHA, is able (i) to reduce the neurotoxic effect of neurotoxin 6-hydroxydopamine (6-OHDA) in vitro, (ii) to re-establish mitochondrial function, and (iii) to increase BNDF and GDNF expression via epigenetic mechanisms.

Effect of EPA on Neonatal Pig Sertoli Cells "In Vitro": A Possible Treatment to Help Maintain Fertility in Pre-Pubertal Boys Undergoing Treatment With Gonado-Toxic Therapies.

The incidence of cancer in pre-pubertal boys has significantly increased and, it has been recognized that the gonado-toxic effect of the cancer treatments may lead to infertility. Here, we have evaluated the effects on porcine neonatal Sertoli cells (SCs) of three commonly used chemotherapy drugs; cisplatin, 4-Hydroperoxycyclophosphamide and doxorubicin. All three drugs induced a statistical reduction of 5-hydroxymethylcytosine in comparison with the control group, performed by Immunofluorescence Analysis. The gene and protein expression levels of GDNF, were significantly down-regulated after treatment to all three chemotherapy drugs comparison with the control group. Specifically, differences in the mRNA levels of GDNF were: 0,8200 ± 0,0440, 0,6400 ± 0,0140, 0,4400 ± 0,0130 fold change at 0.33, 1.66, and 3.33µM cisplatin concentrations, respectively (**p < 0.01 at 0.33 and 1.66 µM vs SCs and ***p < 0.001 at 3.33µM vs SCs); 0,6000 ± 0,0340, 0,4200 ± 0,0130 fold change at 50 and 100 µM of 4-Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7000 ± 0,0340, 0,6200 ± 0,0240, 0,4000 ± 0,0230 fold change at 0.1, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 µM vs SCs and ***p < 0.001 at 1 µM vs SCs). Differences in the protein expression levels of GDNF were: 0,7400 ± 0,0340, 0,2000 ± 0,0240, 0,0400 ± 0,0230 A.U. at 0.33, 1.66, and 3.33µM cisplatin concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,7300 ± 0,0340, 0,4000 ± 0,0130 A.U. at 50 and 100 µM of 4- Hydroperoxycyclophosphamide concentrations, respectively (**p < 0.01 at both these concentrations vs SCs); 0,6200 ± 0,0340, 0,4000 ± 0,0240, 0,3800 ± 0,0230 A.U. at 0.l, 0.2 and 1 µM doxorubicin concentrations, respectively (**p < 0.01 at 0.1 and 0.2 µM vs SCs and ***p < 0.001 at 1 µM vs SCs). Furthermore, we have demonstrated the protective effect of eicosapentaenoic acid on SCs only at the highest concentration of cisplatin, resulting in an increase in both gene and protein expression levels of GDNF (1,3400 ± 0,0280 fold change; **p < 0.01 vs SCs); and of AMH and inhibin B that were significantly recovered with values comparable to the control group. Results from this study, offers the opportunity to develop future therapeutic strategies for male fertility management, especially in pre-pubertal boys.

Molecular mechanisms underlying eicosapentaenoic acid inhibition of HDAC1 and DNMT expression and activity in carcinoma cells.

DNA methylation and histone acetylation, the most studied epigenetic changes, drive and maintain cancer phenotypes. DNA methyltransferase (DNMT) dysregulation promoted localized hypermethylation in CpG rich regions while upregulated histone deacetylases (HDAC) deacetylated histone tails. Both changes led to close chromatin conformation, suppressing transcription and silencing tumor suppressor genes. Consequently, HDAC and DNMT inhibitors appeared to reprogram the transcriptional circuit and potentiate anti-tumoral activity. Here, we report that eicosapentaenoic acid (EPA), a fatty acid with anti-cancer properties, inhibited HDAC1 and DNMT expression and activity, thus promoting tumor suppressor gene expression. In hepatocarcinoma cells (HCC) EPA bound and activated PPARγ thus downregulating HDAC1 which sequentially reduced expression of DNMT1, 3A and 3B. At the same time, activated PPARγ physically interacted with DNMT1 and HDAC1 in a CpG island on the Hic-1 gene to assemble PPARγ/DNMT1 and PPARγ/HDAC1 protein complexes, which exited from DNA. When EPA and PPARγ were no longer bound, the protein complexes separated into individual proteins. Consequently, DNMT1 and HDAC1 down-regulation and release from DNA inhibited their activities. Overall, EPA-bound PPARγ induced re-expression of the tumor suppressor gene Hic-1. In the present study PPARγ emerged as a master regulator acting synergistically through diverse targets and ways to reveal the epigenetic action of EPA as an HDAC1 and DNMT1 inhibitor.

Eicosapentaenoic acid induces DNA demethylation in carcinoma cells through a TET1-dependent mechanism.

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 p21Waf1/Cip1 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.

Blast crisis Ph+ chronic myeloid leukemia with NUP98/HOXA13 up-regulating MSI2.

BACKGROUND: Musashi2(Msi2)-Numb pathway de-regulation is a molecular mechanism underlying the transition of chronic phase Ph + CML to deadly blast crisis, particularly in cases with a NUP98/HOXA9 fusion from a t(7;11)(p15;p15). This study provides new insights on the mechanisms cooperating in driving MSI2 over-expression and progression of Ph-positive CML. RESULTS: Herein we describe a t(7;11)(p15;p15) originating a NUP98 fusion with HOXA13, at 7p15, in a 39 year-old man in blast crisis of Ph-positive CML. Both MSI2 and HOXA9 were evaluated by quantitative RT-PCR in our patient and in a series of haematological malignancies. Up-regulation of both genes emerged only in the presence of NUP98/HOXA13 gene fusion. However, over-expression of MSI2, but not HOXA9, was found in 2 cases of Ph + blast crisis with additional chromosome aberrations other than t(7;11). To determine the mechanisms underlying MSI2 over-expression in our patient we performed Chromatin Immunoprecipitation and found that NUP98/HOXA13 fusion protein deregulates MSI2 gene by binding its promoter. CONCLUSIONS: To the best of our knowledge, this is the first molecular characterization of NUP98/HOXA13 fusion in blast crisis of Ph + CML. Our findings suggest cooperative mechanisms of MSI2 over-expression driven by HOXA proteins and strongly supports MSI2 as a prognostic marker and a candidate in target treatment of CML.

Eicosapentaenoic acid activates RAS/ERK/C/EBPß pathway through H-Ras intron 1 CpG island demethylation in U937 leukemia cells.

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/EBPß/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/EBPß pathway, known to be induced during the monocyte differentiation program. We found that EPA conditioning of U937 leukemia cells activated RAS/ERK/C/EBPß pathway, increasing the C/EBPß 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/EBPß 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.

A diet supplemented with ALA-rich flaxseed prevents cardiomyocyte apoptosis by regulating caveolin-3 expression.

AIMS: n-3 polyunsaturated fatty acids (PUFAs) induce beneficial effects on the heart, but the mechanisms through which these effects are operated are not completely clarified yet. Among others, cardiac diseases are often associated with increased levels of cytokines, such as tumour necrosis factor-α (TNF), that cause degeneration and death of cardiomyocytes. The present study has been carried out to investigate (i) the potential anti-apoptotic effects induced by the n-3 polyunsaturated α-linolenic acid (ALA) in experimental models of cardiac diseases characterized by high levels of TNF, and (ii) the potential role of caveolin-3 (Cav-3) in the mechanisms involved in this process. METHODS AND RESULTS: An ALA-rich flaxseed diet, administered from weaning to hereditary cardiomyopathic hamsters, prevented the onset of myocardial apoptosis associated with high plasma and tissue levels of TNF preserving caveolin-3 expression. To confirm these findings, isolated neonatal mouse cardiomyocytes were exposed to TNF to induce apoptosis. ALA pre-treatment greatly enhanced Cav-3 expression hampering the internalization of the caveolar TNF receptor and, thus, determining the abortion of the apoptotic vs. survival cascade. CONCLUSION: This study unveiled the Cav-3 pivotal role in defending cardiomyocytes against the TNF pro-apoptotic action and the ALA capacity to regulate this mechanism preventing cardiac degenerative diseases.

Adipogenic potential in human mesenchymal stem cells strictly depends on adult or foetal tissue harvest.

Cell-based therapies promise important developments for regenerative medicine purposes. Adipose tissue and the adipogenic process has become central to an increasing number of translational efforts in addition to plastic and reconstructive surgical applications. In recent experimental clinical trials, human mesenchymal stem cells (MSC) have been proven to be well tolerated because of their low immunoreactivity. MSC are multipotent cells found among mature cells in different tissues and organs with the potentiality to differentiate in many cell types, including osteocytes, chondrocytes and adipocytes, thus being a suitable cell source for tissue engineering strategies. We compared the adipogenic potential of MSC originated from two adult sources as fat pads and bone marrow, and from four foetal sources as umbilical cord blood, Wharton's jelly, amniotic fluid and preterm umbilical cord perivascular cells. Surprisingly, adult MSC displayed higher differentiation capacities confirmed by gene expression analysis on a selected panel of adipogenesis-related genes. Further, an in-depth molecular analysis highlighted the early and vigorous activation of the PPARγ transcription factor-cascade in adipose-derived MSC that resulted to be both delayed and reduced in foetal MSC accounting for their lack of adipogenic potential. Thus, MSC show a different degree of phenotypic plasticity depending on the source tissue, that should be taken into consideration for the selection of the most appropriate MSC type for specific tissue regeneration purposes.

Eicosapentaenoic acid demethylates a single CpG that mediates expression of tumor suppressor CCAAT/enhancer-binding protein delta in U937 leukemia cells.

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, α-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/EBPß and C/EBPδ), 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/EBPß and C/EBPδ bind the same response element as a heterodimer. We focused on the enhanced expression of C/EBPδ, 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/EBPδ promoter region, we found a site-specific CpG demethylation that was a determinant for the binding activity of Sp1, an essential factor for C/EBPδ 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/EBPδ is controlled by the methylation state of a site-specific CpG dinucleotide.

Effect of dietary saturated fatty acids on HNF-4α DNA binding activity and ApoCIII mRNA in sedentary rat liver.

Hind limb-suspended rats represent a sedentary-hyperinsulinemic model with a liver dyslipidemia mainly related to changes in sterol regulatory element-binding protein 1 (SREBP-1) and peroxisome proliferator-activated receptor-α (PPARα) expression and activity. To assess the effects of dietary fatty acids on hepatic lipid homeostasis, the hepatic expression and activity of PPARα, SREBP-1, and hepatocyte nuclear factor-4α (HNF-4α) were investigated in this animal model. In control and sedentary rats, diets enriched with saturated, monounsaturated, and polyunsaturated fatty acids (PUFA) enhanced the expression of the PPARα target genes carnitine palmitoyltransferase 1 and acyl-CoA oxidase, the highest effect being exerted by ω-3. The same diets reduced SREBP-1 mRNA and target lipogenic gene expression, as indicated by the reduction in fatty acid synthase and acetyl-CoA carboxylase mRNA content. Effects were greater in sedentary rat liver than in controls on the same diet. Only the ω-3 enriched diet decreased liver triglyceride content as well as plasma cholesterol and triglyceride levels in sedentary rats. This effect may be mainly related to the enhanced mitochondrial and peroxisomal ß-oxidation genes expression. On the other hand, saturated fatty acid-enriched diet induced an increase in liver triglyceride content and enhanced plasma cholesterol and triglyceride levels, both in control and immobilized rats. This detrimental effect may be ascribed to the induced HNF-4α binding activity on ApoCIII promoter and to the enhanced ApoCIII mRNA levels both in control and in sedentary rat livers. In conclusion, we can speculate that dietary saturated fats, acting at apolipoprotein transcriptional level, may impact on the close relationship existing among high ApoCIII plasma level, dyslipidemia, and atherosclerosis.

An omega-3 fatty acid-enriched diet prevents skeletal muscle lesions in a hamster model of dystrophy.

Currently, despite well-known mutational causes, a universal treatment for neuromuscular disorders is still lacking, and current therapeutic efforts are mainly restricted to symptomatic treatments. In the present study, δ-sarcoglycan-null dystrophic hamsters were fed a diet enriched in flaxseed-derived ω3 α-linolenic fatty acid from weaning until death. α-linolenic fatty acid precluded the dystrophic degeneration of muscle morphology and function. In fact, in dystrophic animals fed flaxseed-derived α-linolenic fatty acid, the histological appearance of the muscular tissue was improved, the proliferation of interstitial cells was decreased, and the myogenic differentiation originated new myocytes to repair the injured muscle. In addition, muscle myofibers were larger and cell membrane integrity was preserved, as witnessed by the correct localization of α-, ß-, and γ-sarcoglycans and α-dystroglycan. Furthermore, the cytoplasmic accumulation of both ß-catenin and caveolin-3 was abolished in dystrophic hamster muscle fed α-linolenic fatty acid versus control animals fed standard diet, while α-myosin heavy chain was expressed at nearly physiological levels. These findings, obtained by dietary intervention only, introduce a novel concept that provides evidence that the modulation of the plasmalemma lipid profile could represent an efficacious strategy to ameliorate human muscular dystrophy.

Alpha-linolenic acid-enriched diet prevents myocardial damage and expands longevity in cardiomyopathic hamsters.

Randomized clinical trials have demonstrated that the increased intake of omega-3 polyunsaturated fatty acids significantly reduces the risk of ischemic cardiovascular disease, but no investigations have been performed in hereditary cardiomyopathies with diffusely damaged myocardium. In the present study, delta-sarcoglycan-null cardiomyopathic hamsters were fed from weaning to death with an alpha-linolenic acid (ALA)-enriched versus standard diet. Results demonstrated a great accumulation of ALA and eicosapentaenoic acid and an increased eicosapentaenoic/arachidonic acid ratio in cardiomyopathic hamster hearts, correlating with the preservation of myocardial structure and function. In fact, ALA administration preserved plasmalemma and mitochondrial membrane integrity, thus maintaining proper cell/extracellular matrix contacts and signaling, as well as a normal gene expression profile (myosin heavy chain isoforms, atrial natriuretic peptide, transforming growth factor-beta1) and a limited extension of fibrotic areas within ALA-fed cardiomyopathic hearts. Consequently, hemodynamic indexes were safeguarded, and more than 60% of ALA-fed animals were still alive (mean survival time, 293+/-141.8 days) when all those fed with standard diet were deceased (mean survival time, 175.9+/-56 days). Therefore, the clinically evident beneficial effects of omega-3 polyunsaturated fatty acids are mainly related to preservation of myocardium structure and function and the attenuation of myocardial fibrosis.

Dietary PUFA modulate the expression of proliferation and differentiation markers in Morris 3924A hepatoma cells.

The effect of dietary polyunsaturated fatty acids on the expression of differentiation and proliferation markers in Morris 3924A hepatoma cells was investigated. ACT/I rats were conditioned 10 days with diets enriched with linoleic acid or alpha-linolenic acid before subcutaneous hepatoma cell transplantation. After 19 days from the inoculum, the mRNA levels of liver-enriched transcription factors and of their target genes were quantified. Both linoleic acid- and linolenic acid-enriched diets induced a decrease of beta-actin, AFP, PCNA, c-myc and of hepatocyte nuclear factors HNF-1alpha and HNF-4alpha mRNA levels in tumor tissue whereas HNF-3beta expression was induced by both dietary treatments. Only the alpha-linolenic acid-enriched diet was effective in reducing c-jun and increasing albumin mRNA levels. Since albumin is a C/EBPalpha target gene, C/EBPalpha gene transcription was evaluated at both protein and mRNA levels. It was found that alpha-linolenic acid-enriched diet did not enhance the C/EBPalpha mRNA content in hepatoma tissue while inducing C/EBPalpha protein expression with an isoform pattern similar to the hepatic phenotype. This evidence implies that alpha-linolenic acid or one of its metabolic products induce albumin synthesis in hepatoma cells by modulating C/EBPalpha gene expression at post-transcriptional level.

Identification of a new missense mutation in the mtDNA of hereditary hypertrophic, but not dilated cardiomyopathic hamsters.

The cardiomyopathic hamster is characterized by a naturally occurring deletion in the delta-sarcoglycan gene generating either the hypertrophic or the dilatative phenotype of cardiomyopathy. This evidence suggests that other genetic or environmental factors might concur to the pathogenesis of cardiomyopathy. The aim of the present study was to investigate on the possibility that other genes are involved in the pathogenesis of hamster cardiomyopathy. For this purpose, a series of genes of cardiomyopathic and healthy hamsters were compared by the differential display technique. The hamster cytochrome c oxidase mitochondrial subunit III (COIII) gene has been sequenced and identified as the gene upregulated in brain and skeletal muscle. The gene sequencing and restriction analysis demonstrated that a missense mutation is present in the COIII gene of hamsters exhibiting hypertrophic cardiomyopathy while no mutations were present in dilatative cardiomyopathic hamsters. The mutation was heteroplasmic and the heteroplasmy level was increased with age in skeletal muscle and heart. The ultrastructural analysis of cardiac tissue showed severe damage in the mitochondrial structure of hypertrophic but not dilatative hamster hearts. These results suggest that the pathogenesis of the cardiac damage in hypertrophic cardiomyopathic hamster may be sustained by multiple mutations exerting a cumulative effect on both structure and function of cardiac muscle.