Prostaglandin E3 Research Articles

A Metabolomics Study of Feces Revealed That a Disturbance of Selenium-Centered Metabolic Bioprocess Was Involved in Kashin-Beck Disease, an Osteoarthropathy Endemic to China.

Background: Kashin-Beck disease (KBD) is a distinct osteoarthropathy in China with an unclear pathogenesis. This study aims to explore whether perturbations in the intestine metabolome could be linked to KBD individuals. Methods: An investigation was conducted in KBD endemic villages and fecal samples were collected. After applying inclusion and exclusion criteria, a total of 75 subjects were enrolled for this study, including 46 KBD (including 19 Grade I KBD and 27 Grade II KBD) and 29 controls. Untargeted metabolomics analysis was performed on the platform of UHPLC-MS. PLS-DA and OPLS-DA were conducted to compare the groups and identify the differential metabolites (DMs). Pathway analysis was conducted on MPaLA platform to explore the functional implication of the DMs. Results: Metabolomics analysis showed that compared with the control group, KBD individuals have a total of 584 differential metabolites with dysregulated levels such as adrenic acid (log2FC = -1.87, VIP = 4.84, p = 7.63 × 10-7), hydrogen phosphate (log2FC = -2.57, VIP = 1.27, p = 1.02 × 10-3), taurochenodeoxycholic acid (VIP = 1.16, log2FC = -3.24, p = 0.03), prostaglandin E3 (VIP = 1.17, log2FC = 2.67, p = 5.61 × 10-4), etc. Pathway analysis revealed several significantly perturbed pathways associated with KBD such as selenium micronutrient network (Q value = 3.11 × 10-3, Wikipathways), metabolism of lipids (Q value = 8.43 × 10-4, Reactome), free fatty acid receptors (Q value = 3.99 × 10-3, Reactome), and recycling of bile acids and salts (Q value = 2.98 × 10-3, Reactome). Subgroup comparisons found a total of 267 differential metabolites were shared by KBD vs. control, KBD II vs. control, and KBD I vs. control, while little difference was found between KBD II and KBD I (only one differential metabolite detected). Conclusions: KBD individuals showed distinct metabolic features characterized by perturbations in lipid metabolism and selenium-related bioprocesses. Our findings suggest that the loss of nutrients metabolism balance in intestine was involved in KBD pathogenesis. Linking the nutrients metabolism (especially selenium and lipid) to KBD cartilage damage should be a future direction of KBD study.

Eicosapentaenoic Acid Influences the Lipid Profile of an In Vitro Psoriatic Skin Model Produced with T Cells.

Psoriasis is a skin disease characterized by epidermal hyperplasia and an inappropriate activation of the adaptive immunity. A dysregulation of the skin's lipid mediators is reported in the disease with a predominance of the inflammatory cascade derived from n-6 polyunsaturated fatty acids (n-6 PUFAs). Bioactive lipid mediators derived from arachidonic acid (AA) are involved in the inflammatory functions of T cells in psoriasis, whereas n-3 PUFAs' derivatives are anti-inflammatory metabolites. Here, we sought to evaluate the influence of a supplementation of the culture media with eicosapentaenoic acid (EPA) on the lipid profile of a psoriatic skin model produced with polarized T cells. Healthy and psoriatic skin substitutes were produced following the auto-assembly technique. Psoriatic skin substitutes produced with or without T cells presented increased epidermal and dermal linolenic acid (LA) and AA levels. N-6 PUFA lipid mediators were strongly measured in psoriatic substitutes, namely, 13-hydroxyoctadecadienoic acid (13-HODE), prostaglandin E2 (PGE2) and 12-hydroxyeicosatetraenoic acid (12-HETE). The added EPA elevated the amounts of EPA, n-3 docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) in the epidermal and dermal phospholipids. The EPA supplementation balanced the production of epidermal lipid mediators, with an increase in prostaglandin E3 (PGE3), 12-hydroxyeicosapentaenoic acid (12-HEPE) and N-eicosapentaenoyl-ethanolamine (EPEA) levels. These findings show that EPA modulates the lipid composition of psoriatic skin substitutes by encouraging the return to a cutaneous homeostatic state.

Impact of the TRPV2 Inhibitor on Advanced Heart Failure in Patients with Muscular Dystrophy: Exploratory Study of Biomarkers Related to the Efficacy of Tranilast.

Cardiomyopathy is the leading cause of death in patients with muscular dystrophy (MD). Tranilast, a widely used anti-allergic drug, has displayed inhibitory activity against the transient receptor potential cation channel subfamily V member 2 and improved cardiac function in MD patients. To identify urinary biomarkers that assess improved cardiac function after tranilast administration, we performed a urinary metabolomic study focused on oxidative fatty acids. Accompanying the clinical trial of tranilast, urine specimens were collected over 24 weeks from MD patients with advanced heart failure. Urinary levels of tetranor-PGDM (tetranor-prostaglandin D metabolite), a metabolite of prostaglandin D2, significantly decreased 12 weeks after tranilast administration and were correlated with BNP. These results suggest that prostaglandin-mediated inflammation, which increases with the pathological progression of heart failure in MD patients, was attenuated. Urinary prostaglandin E3 (PGE3) levels significantly increased 4 weeks after tranilast administration. There were positive correlations between the urinary levels of PGE3 and 8-hydroxy-2'-deoxyguanosine, an oxidative stress marker. High PGE3 levels may have a protective effect against cardiomyopathy in MD patients with high oxidative stress. Although further validation studies are necessary, urinary tetranor-PGDM and PGE3 levels may help the current understanding of the extent of advanced heart failure in patients with MD after tranilast administration.

Glucose mediates upregulation of Cck expression independent of cAMP signaling

Cholecystokinin (CCK) is a gut peptide hormone that is also produced by pancreatic β‐cells under conditions of metabolic stress and obesity. Treatment with exogenous CCK can alleviate obesity‐related diabetes in animal models without significant side effects. We have established that CCK is expressed primarily in the pancreatic β‐cell and that CCK is highly upregulated in ob/ob mice. We have also shown that elevated cellular cAMP stimulates β‐cell CCK expression and secretion. However, the specific islet cell types that express CCK and the regulation of Cck transcription in the islet has not been thoroughly explored. A more recent study from our lab showed that high glucose concentrations can upregulate Cck expression in mouse islets and human islets. We hypothesize that Cck expression is upregulated in response to various diabetogenic conditions and that CCK upregulation by glucose can occur independent of cAMP activation.Islets from 16‐week‐old C57BL/6J mice were harvested for use in a dose‐dependent 24‐hour study using different glucose concentrations: No glucose, 2.5mM, 7mM, 11mM & 25mM. After 24‐hour treatment, islets were collected for RNA isolation and cDNA synthesis to identify changes in Cck mRNA expression after incubation with different glucose concentrations. Cck mRNA levels were elevated 4‐8 fold in islets treated with 25 mM glucose compared to islets treated with 0 to 11mM glucose. Notably, the hypoglycemic treatment of no glucose and 2.5mM showed a 4‐fold increase when compared to the 7mM and 11mM treatments. Following this experiment, we wanted to determine if high glucose would still increase Cck mRNA expression after inhibiting cAMP. Islets were pre‐incubated for 45 minutes with 10nM sulprostone, which activated prostaglandin E3 (EP3) receptor and inhibits adenylate cyclase. After inhibition of cAMP, we incubated the islets at 2.5mM and 25mM glucose concentrations like the prior experiment and observed changes in Cck mRNA expression. After treatment with sulprostone, we are still able to see a significant increase in Cck mRNA expression in islets treated with 25mM glucose compared to those treated with 2.5mM glucose, even with cAMP inhibition.This data suggests that there are alternative pathways that could lead to CCK upregulation in the pancreatic islet independent of cAMP. It also shows that hypoglycemia may also increase Cckexpression, which suggests that CCK is upregulated in response to several forms of metabolic stress. We can further explore the effectiveness of glucose to increase CCK expression by treating islets with cycloheximide and deoxyglucose to determine if glucose metabolism is required for upregulation of Cck.This information will help with our general understanding of how CCK functions in pancreatic islet and how Cck transcription is regulated through signaling pathways and transcriptional networks that regulate β‐cell function and identity under diabetogenic conditions.

Sputum microRNA-screening reveals Prostaglandin EP3 receptor as selective target in allergen-specific immunotherapy.

Several microRNAs (miRs) have been described as potential biomarkers in liquid biopsies and in the context of allergic asthma, while therapeutic effects on the airway expression of miRs remain elusive. In this study, we investigated epigenetic miR-associated mechanisms in the sputum of grass pollen-allergic patients with and without allergen-specific immunotherapy (AIT). Induced sputum samples of healthy controls (HC), AIT-treated and -untreated grass pollen-allergic rhinitis patients with (AA) and without asthma (AR) were profiled using miR microarray and whole-transcriptome microarray analysis of the same samples. miR targets were predicted in silico and used to identify inverse regulation. Local PGE2 levels were measured using ELISA. Two hundred and fifty ninemiRs were upregulated in the sputum of AA patients compared with HC, while only one was downregulated. The inverse picture was observed in induced sputum of AIT-treated patients: while 21miRs were downregulated, only 4miRs were upregulated in asthmatics upon AIT. Of these 4miRs, miR-3935stood out, as its predicted target PTGER3, the prostaglandin EP3 receptor, was downregulated in treated AA patients compared with untreated. The levels of its ligand PGE2 in the sputum supernatants of these samples were increased in allergic patients, especially asthmatics, and downregulated after AIT. Finally, local PGE2 levels correlated with ILC2 frequencies, secreted sputum IL-13levels, inflammatory cell load, sputum eosinophils and symptom burden. While profiling the sputum of allergic patients for novel miR expression patterns, we uncovered an association between miR-3935 and its predicted target gene, the prostaglandin E3 receptor, which might mediate AIT effects through suppression of the PGE2 -PTGER3 axis.

Prostaglandin E3 attenuates macrophage-associated inflammation and prostate tumour growth by modulating polarization.

Alternative polarization of macrophages regulates multiple biological processes. While M1‐polarized macrophages generally mediate rapid immune responses, M2‐polarized macrophages induce chronic and mild immune responses. In either case, polyunsaturated fatty acid (PUFA)‐derived lipid mediators act as both products and regulators of macrophages. Prostaglandin E3 (PGE3) is an eicosanoid derived from eicosapentaenoic acid, which is converted by cyclooxygenase, followed by prostaglandin E synthase successively. We found that PGE3 played an anti‐inflammatory role by inhibiting LPS and interferon‐γ‐induced M1 polarization and promoting interleukin‐4‐mediated M2 polarization (M2a). Further, we found that although PGE3 had no direct effect on the growth of prostate cancer cells in vitro, PGE3 could inhibit prostate cancer in vivo in a nude mouse model of neoplasia. Notably, we found that PGE3 significantly inhibited prostate cancer cell growth in a cancer cell‐macrophage co‐culture system. Experimental results showed that PGE3 inhibited the polarization of tumour‐associated M2 macrophages (TAM), consequently producing indirect anti‐tumour activity. Mechanistically, we identified that PGE3 regulated the expression and activation of protein kinase A, which is critical for macrophage polarization. In summary, this study indicates that PGE3 can selectively promote M2a polarization, while inhibiting M1 and TAM polarization, thus exerting an anti‐inflammatory effect and anti‐tumour effect in prostate cancer.

Gestational long-term hypoxia induces metabolomic reprogramming and phenotypic transformations in fetal sheep pulmonary arteries.

Gestational long-term hypoxia increases the risk of myriad diseases in infants including persistent pulmonary hypertension. Similar to humans, fetal lamb lung development is susceptible to long-term intrauterine hypoxia, with structural and functional changes associated with the development of pulmonary hypertension including pulmonary arterial medial wall thickening and dysregulation of arterial reactivity, which culminates in decreased right ventricular output. To further explore the mechanisms associated with hypoxia-induced aberrations in the fetal sheep lung, we examined the premise that metabolomic changes and functional phenotypic transformations occur due to intrauterine, long-term hypoxia. To address this, we performed electron microscopy, Western immunoblotting, calcium imaging, and metabolomic analyses on pulmonary arteries isolated from near-term fetal lambs that had been exposed to low- or high-altitude (3,801 m) hypoxia for the latter 110+ days of gestation. Our results demonstrate that the sarcoplasmic reticulum was swollen with high luminal width and distances to the plasma membrane in the hypoxic group. Hypoxic animals were presented with higher endoplasmic reticulum stress and suppressed calcium storage. Metabolically, hypoxia was associated with lower levels of multiple omega-3 polyunsaturated fatty acids and derived lipid mediators (e.g., eicosapentaenoic acid, docosahexaenoic acid, α-linolenic acid, 5-hydroxyeicosapentaenoic acid (5-HEPE), 12-HEPE, 15-HEPE, prostaglandin E3, and 19(20)-epoxy docosapentaenoic acid) and higher levels of some omega-6 metabolites (P < 0.02) including 15-keto prostaglandin E2 and linoleoylglycerol. Collectively, the results reveal broad evidence for long-term hypoxia-induced metabolic reprogramming and phenotypic transformations in the pulmonary arteries of fetal sheep, conditions that likely contribute to the development of persistent pulmonary hypertension.

Dual Behavior of Long-Chain Fatty Acids and Their Cyclooxygenase/Lipoxygenase Metabolites on Human Intestinal Caco-2 Cell Growth.

Etiology of colorectal cancer (CRC) is related, at least in part, with nutritional profile and epidemiological data indicating a key role of dietary fat on CRC pathogenesis. Moreover, inflammation and eicosanoids produced from arachidonic acid might have a pivotal role in CRC development. However, the effect of specific fatty acids (FAs) on intestinal epithelial cell growth is not completely studied now. By this reason, the aim of this work is to unravel the effect of different saturated and unsaturated long-chain fatty acids (LCFA) and some LCFA metabolites on CRC cell line growth and their possible mechanisms of action. Our results demonstrated that oleic acid is a potent mitogenic factor to Caco-2 cells, at least in part, through 10-hydroxy-8-octadecenoic synthesized by lipoxigenase pathway, whereas polyunsaturated FAs such as eicosapentaenoic (EPA) acid has a dual behavior effect depending on its concentration. A high concentration, EPA induced apoptosis through intrinsic pathway, whereas at low concentration induced cell proliferation that could be related to the synthesis of eicosanoids such as prostaglandin E3 and 12-hydroxyeicosapentaenoic acid and the subsequent induction of mitogenic cell signaling pathways (ERK 1/2, CREB, p38α). Thus, this study contributes to understand the complicated relationship between fat ingest and CRC.

Network pharmacology combined with metabolomics approach to investigate the protective role and detoxification mechanism of Yunnan Baiyao formulation

BackgroundYunnan Baiyao (YNBY) is a traditional Chinese medicine formulae, which has the functions of hemostasis, activating blood circulation and removing blood stasis, anti-inflammation, etc. Although the presence of Caowu (CW, Aconiti Kusnezoffii Radix), the detoxification mechanism of YNBY is still unclear. PurposeIn current study, network pharmacology, toxicological methods and metabolomics technique were applied to explore YNBY in attenuating toxicity of CW. MethodsPrediction of targets and pathways of CW were carried out by commonly used network pharmacological method. Simultaneously, SD rats were orally administrated with CW, processed CW (ZCW), YNBY, and YNBY which lack of CW (QCW) for 15 days. Tissue samples were observed with histopathology. Urine samples were analyzed with ultra-performance liquid chromatography-mass spectrometry to screen differential metabolites and related metabolic pathways associated with toxicity of CW. Furthermore, by comparing the changes of the metabolite contents, focused the attenuated metabolic pathway. Finally, the network pharmacological and experimental data were integrated to investigate detoxification mechanism of YNBY. ResultsA total of 44 potential toxicity biomarkers were identified and 14 related pathways were involved in the toxicity of CW. Furthermore, 5 core toxicity biomarkers (2-keto-6-acetamidocaproate, γ-glutamylleucine, prostaglandin E3, 4-hydroxy-5-(3’-hydroxyphenyl)-valeric acid-3’-O-sulphate, and 3,4-dihydroxy- phenylglycol O-sulfate) were regulated to normal condition in YNBY group. Lysine degradation was locked as the core metabolic pathway of detoxification of YNBY. Integrating the predicted results of network pharmacology, ACHE, SLC6A3, SLC6A4 might be the target of protective role of other herbs in YNBY. ConclusionNetwork pharmacology combined with metabolomics exhibited a powerful mean to investigate the herbal toxicity and probed into the detoxification mechanism of formulae, which contributes to its safety evaluation.

Searching integrated strategies for the evaluation of the physiological status in fish fed functional diets: The example of SDPP in gilthead sea bream (Sparus aurata)

In this study we evaluated the effects of high dietary arachidonic acid (ARA) levels on prostaglandin E2 (PGE2) and E3 (PGE3) production and their effect on two morphogenetic processes occurring during metamorphosis: the establishment of the juvenile pigmentation pattern and eye migration and remodeling of cranial bones. In this sense, Senegalese sole larvae were fed from 2 to 50 days post-hatch (dph) with live prey enriched with an experimental emulsion containing high levels of ARA (ARA-H; 10.2 and 7.1% TFA in enriched rotifer and Artemia, respectively) versus a reference commercial enriching product (Algamac 3050®, AGM; 1.0 and 1.4% TFA in enriched rotifer and Artemia, respectively). High dietary ARA levels did not affect larval growth performance at 50 dph, but significantly induced malpigmentation (81.4 ± 7.5%, versus 0.9 ± 0.3% in larvae fed the AGM diet). This malpigmentation was linked to the higher prostaglandin E2 (PGE2) levels observed in pseudo-albino fish as compared to normally pigmented individuals. The PGE2 levels were higher in normally pigmented specimens fed the ARA-H diet than in those fed the AGM diet. The effects of ARA on normally pigmented fish fed the AGM diet and pseudo-albino specimens fed the ARA-H diet were evaluated by means of the density of melanophores and the texture and image segmentation analyses in the dorsal skin of post-metamorphic fish. The skin of pseudo-albino specimens had a more uniform and homogeneous melanophore pattern than normally pigmented fish. Melanophores in pseudo-albino specimens were less abundant and not so aggregated in patches as they were in normally pigmented ones, whereas their shape differed (round vs. dendritic) suggesting their inability to disperse melanin. In addition, fish fed the ARA-H diet presented a higher percentage of cranial deformities (95.1 ± 1.5%) than those fed the control diet (1.9 ± 1.9%) that was significantly and negatively correlated with the incidence of normally-pigmented animals (R2 = − 0.88, P < 0.001). Cranial deformities in pseudo-albino fish were associated with an impaired migration of the eye from the ocular side (the right eye), whereas the left eye migrated from the blind side into the ocular side almost normally. The effects of high dietary ARA levels in the eye migration and cranial bone remodeling processes in post-metamorphic larvae were evaluated by means of the staining of cranial skeletal elements. Pseudo-albino fish showed higher interocular distance and head height than normally pigmented individuals, a different disposition of the eyes with regard to the vertebral column and mouth axes, and a distinct osteological development of some skeletal structures from the neuro- and splanchnocranium, in relation to high dietary ARA contents and high PGE2 production. These results brought new information about possible nutritional forcing factors and physiological mechanisms of pigmentary disorders and impaired eye migration, which are current major bottlenecks in flatfish aquaculture.

Mass spectrometry-based urinary metabolomics for the investigation on the mechanism of action of Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. leaves against ischemic stroke in rats

Ethnopharmacological relevanceAs a traditional Chinese medicine, Eleutherococcus senticosus (Rupr. & Maxim.) Maxim. leaves (ESL) can treat ischemic, neurasthenia, and hypertension diseases. However, only few studies have been conducted on the mechanism of action of ESL for ischemic disease treatment. Aim of the studyThis study aimed to discover the potential biomarkers in the rats caused by ischemic stroke and build a gene–enzyme–biomarker network to explore the mechanism of ESL treatment on ischemic stroke further. Materials and methodsThe urinary metabolomics strategy was developed by combining UPLC-Q-TOF/MS with multivariate data analysis. The gene-enzyme-biomarker network was built by Cytoscape 3.6.0 on the basis of the potential biomarkers filtered out via urinary metabolomic analysis. Then, the potential target enzymes of ESL in the treatment of ischemic stroke were selected for further validation analysis via the ELISA kits. ResultsA total of 42 biomarkers associated with ischemic stroke have been identified, among which 38 species can be adjusted by ESL, including 5′-methylthioadenosine, prostaglandin A2, l-methionine, aldosterone, 11b-hydroxyprogesterone, prostaglandin E3, dehydroepiandrosterone, taurine, 5-methoxyindoleacetate, and p-cresol glucuronide. These biomarkers were involved in several metabolic pathways, including taurine and hypotaurine, arachidonic acid, cysteine and methionine, steroid hormone biosynthesis, tryptophan, and tyrosine metabolism pathways. The gene–enzyme–biomarker network was built, and three predicted target proteins, including cyclooxygenase-2 (COX-2), monoamine oxidase (MAO), and nitric oxide synthase (NOS), were selected as the potential target enzymes for ESL in ischemic stroke treatment. ConclusionsAll results showed that ESL can play a therapeutic role in treating ischemic stroke through different pathways. This study will provide an overall view of the mechanism underlying the action of ESL against ischemic stroke.

An LC–MS-based lipidomics approach for studying the impact of dietary eicosapentaenoic acid on modulating methylmercury toxicity in mice

Methylmercury (MeHg) exposure has been a public health problem for many decades. There is a growing interest in searching for possible dietary nutrients that may affect MeHg toxicity. The study aims to evaluate the impact of eicosapentaenoic acid (EPA) on modulating MeHg toxicity in mice. The study was based on a two-level factorial design, where the factors were presence or absence of EPA and MeHg in the feed. A liquid chromatography-mass spectrometry-based lipidomics approach was used to identify and quantify the main phospholipid species in mouse liver and plasma. The effects of EPA and MeHg on phospholipid species were evaluated by principal component analysis and statistics. Some biochemical and toxicological markers were measured and hepatic histological assay was carried out. EPA treatment significantly elevated the phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) species that contain n-3 PUFA, and reduced the levels of PtdCho and PtdEtn species that contain arachidonic acid (ARA), while MeHg exhibited opposite effects on these specific PtdCho species in liver. MeHg induced higher prostaglandin E2 and lower prostaglandin E3, thus increasing pro-inflammatory factors, while EPA decreased these ARA-derived inflammatory factors. Moreover, MeHg induced chronic inflammatory symptoms in mice, including severe hepatic necrosis, higher aspartate aminotransferase and alanine aminotransferase activities in plasma, higher thiobarbituric acid reactive substances and lower glutathione in liver. These symptoms were all alleviated by EPA treatment. EPA may have protective effect against MeHg-induced toxicity due to the favorable modification of membrane phospholipid composition and inhibition of inflammatory factors.

Prostaglandin transporter (OATP2A1/SLCO2A1) contributes to local disposition of eicosapentaenoic acid-derived PGE3

Eicosapentaenoic acid (EPA)-derived prostaglandin E3 (PGE3) possesses an anti-inflammatory effect; however, information for transporters that regulate its peri-cellular concentration is limited. The present study, therefore, aimed to clarify transporters involved in local disposition of PGE3. PGE3 uptake was assessed in HEK293 cells transfected with OATP2A1/SLCO2A1, OATP1B1/SLCO1B1, OATP2B1/SLCO2B1, OAT1/SLC22A6, OCT1/SLC22A1 or OCT2/SLC22A2 genes, compared with HEK293 cells transfected with plasmid vector alone (Mock). PGE3 uptake by OATP2A1-expressing HEK293 cells (HEK/2A1) was the highest and followed by HEK/1B1, while no significantly higher uptake of PGE3 than Mock cells was detected by other transporters. Saturation kinetics in PGE3 uptake by HEK/2A1 estimated the Km as 7.202±0.595μM, which was 22 times higher than that of PGE2 (Km=0.331±0.131μM). Furthermore, tissue disposition of PGE3 was examined in wild-type (WT) and Slco2a1-deficient (Slco2a1−/−) mice after oral administration of EPA ethyl ester (EPA-E) when they underwent intraperitoneal injection of endotoxin (e.g., lipopolysaccharide). PGE3 concentration was significantly higher in the lung, and tended to increase in the colon, stomach, and kidney of Slco2a1−/−, compared to WT mice. Ratio of PGE2 metabolite 15-keto PGE2 over PGE2 concentration was significantly lower in the lung and colon of Slco2a1−/− than that of WT mice, suggesting that PGE3 metabolism is downregulated in Slco2a1−/− mice. In conclusion, PGE3 was found to be a substrate of OATP2A1, and local disposition of PGE3 could be regulated by OATP2A1 at least in the lung.

Effects of a Mediterranean Diet Intervention on Anti- and Pro-Inflammatory Eicosanoids, Epithelial Proliferation, and Nuclear Morphology in Biopsies of Normal Colon Tissue

This randomized trial evaluated the effects of intervention with either a Healthy Eating or a Mediterranean diet on colon biomarkers in 120 healthy individuals at increased colon cancer risk. The hypothesis was that eicosanoids and markers of proliferation would be favorably affected by the Mediterranean diet. Colon epithelial biopsy tissues and blood samples were obtained at baseline and after 6 mo of intervention. Colonic eicosanoid concentrations were evaluated by HPLC-MS-MS, and measures of epithelial proliferation and nuclear morphology were evaluated by image analysis of biopsy sections. There was little change in proinflammatory eicosanoids and in plasma cytokine concentrations with either dietary intervention. There was, however, a 50% increase in colonic prostaglandin E3 (PGE3), which is formed from eicosapentanoic acid, in the Mediterranean arm. Unlike PGE2, PGE3, was not significantly affected by regular use of non-steroidal anti-inflammatory drugs at baseline, and normal weight subjects had significantly higher colon PGE3 than overweight or obese subjects. Increased proliferation in the colon at baseline, by Ki67 labeling, was associated with morphological features that defined smaller nuclei in the epithelial cells, lower colon leukotriene concentrations and higher plasma cytokine concentrations. Dietary intervention had little effect on measures of epithelial proliferation or of nuclear morphology. The increase in PGE3 with a Mediterranean diet indicates that in normal colon, diet might affect protective pathways to a greater extent than proinflammatory and proliferative pathways. Hence, biomarkers from cancer models might not be relevant in a true prevention setting.

Transport of eicosapentaenoic acid-derived PGE₃, PGF(3α), and TXB₃ by ABCC4.

BackgroundEicosapentaenoic acid-derived prostaglandin (PG) E3, PGF3α, and thromboxane (TX) B3 are bioactive lipid mediators which have anti-cancer and anti-inflammatory effects. To exert their effects, PGE3, PGF3α, and TXB3 must be released to the extracellular space from cells, but the release mechanism has been unclear. We therefore investigated the contribution of ATP-binding cassette transporter C4 (ABCC4), which has been known as a prostanoids efflux transporter, to the release of PGE3, PGF3α, and TXB3.Materials and MethodsATP-dependent transport of PGE3, PGF3α, and TXB3 via ABCC4 was investigated by using inside-out membrane vesicles prepared from ABCC4-overexpressing HEK293 cells. To evaluate the contribution of ABCC4 to the release of PGE3, PGF3α, and TXB3, we measured the extracellular and intracellular levels of PGE3, PGF3α, and TXB3 in A549 cells when we used ABCC4 inhibitors (dipyridamole, MK571, and probenecid) or ABCC4 siRNAs. The quantification of PGE3, PGF3α, and TXB3 was performed by using liquid chromatography-tandem mass spectrometry.ResultsThe apparent Km values for ABCC4-mediated transport were 2.9±0.1 µM for PGE3, 12.1±1.3 µM for PGF3α, and 11.9±1.4 µM for TXB3 and the ATP-dependent accumulation of PGE3, PGF3α, and TXB3 into vesicles was decreased by using typical substrates and inhibitors of ABCC4. ABCC4 inhibitors and ABCC4 knockdown showed the reduction of extracellular/intracellular ratio of PGE3 (40–60% of control) and PGF3α (60–80% of control) in A549 cells.ConclusionsOur results suggest that PGE3, PGF3α, and TXB3 are substrates of ABCC4 and ABCC4 partially contributes to the release of PGE3 and PGF3α.

Quantification of intracellular and extracellular eicosapentaenoic acid-derived 3-series prostanoids by liquid chromatography/electrospray ionization tandem mass spectrometry

3-Series prostanoids are bioactive lipid mediators synthesized from eicosapentaenoic acid (EPA). Determination of intracellular and extracellular levels of prostanoids is needed to elucidate the mechanism of action, and we therefore developed a method for quantification of intracellular and extracellular levels of 3-series prostanoids (including prostaglandin E3 (PGE3), PGD3, PGF3α, thromboxane B3 (TXB3), and Δ17-6-keto PGF1α) by using liquid chromatography/electrospray ionization tandem mass spectrometry. The separation of prostanoids was performed with a CAPCELL PAK C18 MG II column (2.0mm×150mm, 3µm) with an isocratic flow of acetonitrile/water/acetic acid (40:60:0.1, v/v/v). This method was validated for measurement of both extracellular and intracellular samples with high levels of precision and accuracy. We applied this method to human lung epithelial A549 cells stimulated with calcium ionophore A23187 under the condition of arachidonic acid or EPA treatment and we could measure PGE3 in both intracellular and extracellular samples.

Inhibition of the HER2 pathway by n-3 polyunsaturated fatty acids prevents breast cancer in fat-1 transgenic mice

Overexpression of the tyrosine kinase receptor, ErbB2/HER2/Neu, occurs in 25-30% of invasive breast cancer (BC) with poor patient prognosis. Due to confounding factors, inconsistencies still remain regarding the protective effects of n-3 polyunsaturated fatty acids (PUFAs) on BC. We therefore evaluated whether fat-1 transgenic mice, endogenously synthesizing n-3 PUFAs from n-6 PUFAs, were protected against BC development, and we then aimed to study in vivo a mechanism potentially involved in such protection. E0771 BC cells were implanted into fat-1 and wild-type (WT) mice. After tumorigenesis examination, we analyzed the expression of proteins involved in the HER2 signaling pathway and lipidomic analyses were performed in tumor tissues and plasma. Our results showed that tumors totally disappeared by day 15 in fat-1 mice but continued to grow in WT mice. This prevention can be related in part to significant repression of the HER2/β-catenin signaling pathway and formation of significant levels of n-3 PUFA-derived bioactive mediators (particularly 15-hydroxyeicosapentaenoic acid, 17-hydroxydocosahexaenoic acid, and prostaglandin E3) in the tumors of fat-1 mice compared with WT mice. All together these data demonstrate an anti-BC effect of n-3 PUFAs through, at least in part, HER2 signaling pathway downregulation, and highlight the importance of gene-diet interactions in BC.

Changes in Cancer Cell Metabolism Revealed by Direct Sample Analysis with MALDI Mass Spectrometry

Biomarker discovery using mass spectrometry (MS) has recently seen a significant increase in applications, mainly driven by the rapidly advancing field of metabolomics. Instrumental and data handling advancements have allowed for untargeted metabolite analyses which simultaneously interrogate multiple biochemical pathways to elucidate disease phenotypes and therapeutic mechanisms. Although most MS-based metabolomic approaches are coupled with liquid chromatography, a few recently published studies used matrix-assisted laser desorption (MALDI), allowing for rapid and direct sample analysis with minimal sample preparation. We and others have reported that prostaglandin E3 (PGE3), derived from COX-2 metabolism of the omega-3 fatty acid eicosapentaenoic acid (EPA), inhibited the proliferation of human lung, colon and pancreatic cancer cells. However, how PGE3 metabolism is regulated in cancer cells, particularly human non-small cell lung cancer (NSCLC) cells, is not fully understood. Here, we successfully used MALDI to identify differences in lipid metabolism between two human non-small-cell lung cancer (NSCLC) cell lines, A549 and H596, which could contribute to their differential response to EPA treatment. Analysis by MALDI-MS showed that the level of EPA incorporated into phospholipids in H596 cells was 4-fold higher than A549 cells. Intriguingly, H596 cells produced much less PGE3 than A549 cells even though the expression of COX-2 was similar in these two cell lines. This appears to be due to the relatively lower expression of cytosolic phospholipase A2 (cPLA2) in H596 cells than that of A549 cells. Additionally, the MALDI-MS approach was successfully used on tumor tissue extracts from a K-ras transgenic mouse model of lung cancer to enhance our understanding of the mechanism of action of EPA in the in vivo model. These results highlight the utility of combining a metabolomics workflow with MALDI-MS to identify the biomarkers that may regulate the metabolism of omega-3 fatty acids and ultimately affect their therapeutic potentials.

Emerging importance of omega-3 fatty acids in the innate immune response: molecular mechanisms and lipidomic strategies for their analysis.

The beneficial health properties of dietary omega-3 polyunsaturated fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have long been known and their metabolic dysfunction has been linked to a range of diseases including various inflammatory disorders, cardiovascular diseases, and cancer. However, the molecular mechanisms underlying their health benefits have remained unclear. Recent technological advances in lipidomic analytical strategies have resulted in the discovery of a range of bioactive mediators derived from EPA and DHA that possess potent anti-inflammatory and pro-resolving properties and that may be responsible, at least in part, for the beneficial effects observed. These mediators include resolvins, protectins and maresins, as well as EPA derivatives of classical arachidonic acid derived eicosanoids, such as prostaglandin E3 . The aim of this review is to provide an overview of the biosynthetic pathways and biological properties of these omega-3 mediators, with a particular focus on the emerging importance of the counter-regulatory role of omega-3 and -6 fatty acids in the spatial and temporal regulation of the inflammatory response. It will also provide an insight into a range of lipidomic approaches, which are currently available to analyse these fatty acids and their metabolites in biological matrices.

Anticancer activity of fish oils against human lung cancer is associated with changes in formation of PGE2 and PGE3 and alteration of Akt phosphorylation

The beneficial effects of omega-3 fatty acids are believed to be due in part to selective alteration of arachidonate metabolism that involves cyclooxygenase (COX) enzymes. Here we investigated the effect of eicosapentaenoic acid (EPA) on the proliferation of human non-small cell lung cancer A549 (COX-2 over-expressing) and H1299 (COX-2 null) cells as well as their xenograft models. While EPA inhibited 50% of proliferation of A549 cells at 6.05 µM, almost 80 µM of EPA was needed to reach similar levels of inhibition of H1299 cells. The formation of prostaglandin (PG)E3 in A549 cells was almost threefold higher than that of H1299 cells when these cells were treated with EPA (25 µM). Intriguingly, when COX-2 expression was reduced by siRNA or shRNA in A549 cells, the antiproliferative activity of EPA was reduced substantially compared to that of control siRNA or shRNA transfected A549 cells. In line with this, dietary menhaden oil significantly inhibited the growth of A549 tumors by reducing tumor weight by 58.8 ± 7.4%. In contrast, a similar diet did not suppress the development of H1299 xenograft. Interestingly, the ratio of PGE3 to PGE2 in A549 was about 0.16 versus only 0.06 in H1299 xenograft tissues. Furthermore, PGE2 up-regulated expression of pAkt, whereas PGE3 downregulated expression of pAkt in A549 cells. Taken together, the results of our study suggest that the ability of EPA to generate PGE3 through the COX-2 enzyme might be critical for EPA-mediated tumor growth inhibition which is at least partly due to down-regulation of Akt phosphorylation by PGE3.