Docosahexaenoic acid (DHA) promotes immunogenic apoptosis in human multiple myeloma cells, induces autophagy and inhibits STAT3 in both tumor and dendritic cells

Docosahexaenoic acid (DHA), a ω-3 polyunsaturated fatty acid found in fish oil, is a multi-target agent and exerts anti-inflammatory and anticancer activities alone or in combination with chemotherapies. Combinatorial anticancer therapies, which induce immunogenic apoptosis, autophagy and STAT3 inhibition have been proposed for long-term therapeutic success. Here, we found that DHA promoted immunogenic apoptosis in multiple myeloma (MM) cells, with no toxicity on PBMCs and DCs. Immunogenic apoptosis was shown by the emission of specific DAMPs (CRT, HSP90, HMGB1) by apoptotic MM cells and the activation of their pro-apoptotic autophagy. Moreover, immunogenic apoptosis was directly shown by the activation of DCs by DHA-induced apoptotic MM cells. Furthermore, we provided the first evidence that DHA activated autophagy in PBMCs and DCs, thus potentially acting as immune stimulator and enhancing processing and presentation of tumor antigens by DCs. Finally, we found that DHA inhibited STAT3 in MM cells. STAT3 pathway, essential for MM survival, contributed to cancer cell apoptosis by DHA. We also found that DHA inhibited STAT3 in blood immune cells and counteracted STAT3 activation by tumor cell-released factors in PBMCs and DCs, suggesting the potential enhancement of the anti-tumor function of multiple immune cells and, in particular, that of DCs

SWATH Differential Abundance Proteomics and Cellular Assays Show In Vitro Anticancer Activity of Arachidonic Acid- and Docosahexaenoic Acid-Based Monoacylglycerols in HT-29 Colorectal Cancer Cells

Colorectal cancer (CRC) is one of the most common and mortal types of cancer. There is increasing evidence that some polyunsaturated fatty acids (PUFAs) exercise specific inhibitory actions on cancer cells through different mechanisms, as a previous study on CRC cells demonstrated for two very long-chain PUFA. These were docosahexaenoic acid (DHA, 22:6n3) and arachidonic acid (ARA, 20:4n6) in the free fatty acid (FFA) form. In this work, similar design and technology have been used to investigate the actions of both DHA and ARA as monoacylglycerol (MAG) molecules, and results have been compared with those obtained using the corresponding FFA. Cell assays revealed that ARA- and DHA-MAG exercised dose- and time-dependent antiproliferative actions, with DHA-MAG acting on cancer cells more efficiently than ARA-MAG. Sequential window acquisition of all theoretical mass spectra (SWATH)—mass spectrometry massive quantitative proteomics, validated by parallel reaction monitoring and followed by pathway analysis, revealed that DHA-MAG had a massive effect in the proteasome complex, while the ARA-MAG main effect was related to DNA replication. Prostaglandin synthesis also resulted as inhibited by DHA-MAG. Results clearly demonstrated the ability of both ARA- and DHA-MAG to induce cell death in colon cancer cells, which suggests a direct relationship between chemical structure and antitumoral actions

Liability of a Surgeon for the Extension of an Authorized Operation

Polyunsaturated fatty acids modulate the voltage dependence of several voltage-gated ion channels, thereby being potent modifiers of cellular excitability. Detailed knowledge of this molecular mechanism can be used in designing a new class of small-molecule compounds against hyperexcitability diseases. Here, we show that arginines on one side of the helical K-channel voltage sensor S4 increased the sensitivity to docosahexaenoic acid (DHA), whereas arginines on the opposing side decreased this sensitivity. Glutamates had opposite effects. In addition, a positively charged DHA-like molecule, arachidonyl amine, had opposite effects to the negatively charged DHA. This suggests that S4 rotates to open the channel and that DHA electrostatically affects this rotation. A channel with arginines in positions 356, 359, and 362 was extremely sensitive to DHA: 70 mu M DHA at pH 9.0 increased the current greater than500 times at negative voltages compared with wild type (WT). The small-molecule compound pimaric acid, a novel Shaker channel opener, opened the WT channel. The 356R/359R/362R channel drastically increased this effect, suggesting it to be instrumental in future drug screening

Effects of diet and/or exercise in enhancing spinal cord sensorimotor learning.

Given that the spinal cord is capable of learning sensorimotor tasks and that dietary interventions can influence learning involving supraspinal centers, we asked whether the presence of omega-3 fatty acid docosahexaenoic acid (DHA) and the curry spice curcumin (Cur) by themselves or in combination with voluntary exercise could affect spinal cord learning in adult spinal mice. Using an instrumental learning paradigm to assess spinal learning we observed that mice fed a diet containing DHA/Cur performed better in the spinal learning paradigm than mice fed a diet deficient in DHA/Cur. The enhanced performance was accompanied by increases in the mRNA levels of molecular markers of learning, i.e., BDNF, CREB, CaMKII, and syntaxin 3. Concurrent exposure to exercise was complementary to the dietary treatment effects on spinal learning. The diet containing DHA/Cur resulted in higher levels of DHA and lower levels of omega-6 fatty acid arachidonic acid (AA) in the spinal cord than the diet deficient in DHA/Cur. The level of spinal learning was inversely related to the ratio of AA:DHA. These results emphasize the capacity of select dietary factors and exercise to foster spinal cord learning. Given the non-invasiveness and safety of the modulation of diet and exercise, these interventions should be considered in light of their potential to enhance relearning of sensorimotor tasks during rehabilitative training paradigms after a spinal cord injury

The Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) on Brown Adipogenesis in Stem Cell Culture

Polyunsaturated fatty acids (PUFAs), especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are major maternal dietary supplements due to their positive benefits on neurological tissue growth during the first 12 weeks of gestation. Previous studies show that EPA and DHA inhibit muscle formation but promote adipogenesis. However, no research has addressed the question whether high intake of EPA and DHA affects brown fat development during gestation. The objective of this study was to measure the effect of EPA and DHA supplement on brown adipogenesis and potential pathways related to mitochondrial biosynthesis using fibroblasts as in vitro model. Using Oil-Red-O staining and PCR testing, lipid droplet formation and tested six genes were examined and PGC1α presented statistically significant difference from the control group when treated with PUFAs. Results indicated that PGC1α gene expression can be to be alternated by EPA and DHA treatment. Mitochondrial biosynthesis can potentially be promoted by increased PGC1α gene expression. However, the lipid droplets accumulated in the PUFAs treated group show an unknown mechanism of the n-3 PUFA on adipogenesis that needs to be revealed

The impact of fish oil fatty-acids on post-prandial vascular reactivity.

Progressive loss of vascular reactivity and increased vascular tone with age are being increasingly recognised as significant cardiovascular disease (CVD) risk factors. The vasculature has emerged as a target for dietary strategies to modify these progressions. Our previous data suggest that inclusion of fish oil in a high-fat test meal improves postprandial vascular reactivity in healthy men. The primary aim of this project was to determine the individual effects of the fish oil fatty acids, eicosapentaenoic acid (EPA) versus docosahexaenoic acid (DHA) on post-prandial vascular reactivity and to identify underlying molecular mechanisms of these effects. In this study, the acute effects of a single dose (4.16g) of EPA, DHA and placebo oil on postprandial vascular reactivity were determined in men categorised as being at a 1.5 relative risk of CVD in a double-blind randomised crossover trial. Measures of vascular tone and function, alongside an endothelium dependent measure of vascular reactivity, were taken along with blood samples, at baseline and at 4 hours in order to coincide with the anticipated peak plasma concentration of these fatty acids. These blood samples were utilised for assessment of biomarkers associated with changes in vascular tone and postprandial whole blood culture (WBC). WBC was conducted to investigate the effect of changes in the postprandial lipidome, dependent on EPA and DHA consumption, on cytokine production. Response to intervention according to genotype was also determined retrospectively. We observed that DHA (p=0.04) but not EPA (p=0.06) significantly reduced Augmentation Index (AI) postprandially when compared to control. AI was reduced by 13.3% and 11.3% in response to DHA and EPA respectively. In addition, our data shows for the first time, that levels of the vasoactive n-3 PUFA epoxide and diol metabolites are subject to large changes post consumption of physiological levels of EPA and DHA. Our data also suggests there is wide inter-individual variability in circulating levels of these compounds, which may in part explain inter-individual responsiveness to EPA and DHA. We were not able to observe changes in plasma nitrite or H2S levels postprandially, suggesting that EPA and DHA dependent changes in vascular tone may be mediated in part by their vasoactive epoxide and diol metabolites. However, whole Blood Culture experiments did not show a significant effect on any of the cytokines or growth factors investigated, before or after correction for BMI and AGE. Finally, our investigations of response to treatment by genotype suggested a novel interaction between the PPAR-ƴ rs1801282 polymorphism, DHA consumption and improvements in postprandial triglyceridemia. The findings of this thesis emphasise the differential effects of EPA and DHA in the vasculature and the inter-individual responsiveness to these nutrients

Association between high dietary intake of the n-3 polyunsaturated fatty acid docosahexaenoic acid and reduced risk of Crohn's disease

Background: There are plausible mechanisms for how dietary docosahexaenoic acid (DHA), an n−3 polyunsaturated fatty acid, could prevent Crohn's disease (CD). Aim: To conduct a prospective study to investigate the association between increased intake of DHA and risk of CD. Methods: Overall, 229 702 participants were recruited from nine European centres between 1991 and 1998. At recruitment, dietary intakes of DHA and fatty acids were measured using validated food frequency questionnaires. The cohort was monitored through to June 2004 to identify participants who developed incident CD. In a nested case–control analysis, each case was matched with four controls; odds ratios (ORs) were calculated for quintiles of DHA intake, adjusted for total energy intake, smoking, other dietary fatty acids, dietary vitamin D and body mass index. Results: Seventy-three participants developed incident CD. All higher quintiles of DHA intake were inversely associated with development of CD; the highest quintile had the greatest effect size (OR = 0.07; 95% CI = 0.02–0.81). The OR trend across quintiles of DHA was 0.54 (95% CI = 0.30–0.99, Ptrend = 0.04). Including BMI in the multivariate analysis, due to its correlation with dietary fat showed similar associations. There were no associations with the other dietary fatty acids studied. Conclusion: There were inverse associations, with a biological gradient between increasing dietary docosahexaenoic acid intakes and incident Crohn's disease. Further studies in other populations should measure docosahexaenoic acid to determine if the association is consistent and the hypothesis tested in randomised controlled trials of purely docosahexaenoic acid supplementation

Lipoprotein lipase regulates hematopoietic stem progenitor cell maintenance through DHA supply.

Lipoprotein lipase (LPL) mediates hydrolysis of triglycerides (TGs) to supply free fatty acids (FFAs) to tissues. Here, we show that LPL activity is also required for hematopoietic stem progenitor cell (HSPC) maintenance. Knockout of Lpl or its obligatory cofactor Apoc2 results in significantly reduced HSPC expansion during definitive hematopoiesis in zebrafish. A human APOC2 mimetic peptide or the human very low-density lipoprotein, which carries APOC2, rescues the phenotype in apoc2 but not in lpl mutant zebrafish. Creating parabiotic apoc2 and lpl mutant zebrafish rescues the hematopoietic defect in both. Docosahexaenoic acid (DHA) is identified as an important factor in HSPC expansion. FFA-DHA, but not TG-DHA, rescues the HSPC defects in apoc2 and lpl mutant zebrafish. Reduced blood cell counts are also observed in Apoc2 mutant mice at the time of weaning. These results indicate that LPL-mediated release of the essential fatty acid DHA regulates HSPC expansion and definitive hematopoiesis

N-3 Polyunsaturated Fatty Acids (PUFAs) Reverse the Impact of Early-Life Stress on the Gut Microbiota

Supporting Information S1 File. Microbiota Data Set. NS.S, NS.LD, NS.HD stand for non-separated Saline, non-separated Low Dose, non-separated High Dose, respectively. MS.S, MS.LD, MS.HD stand for maternally separated Saline, maternally separated Low Dose, maternally separated High Dose, respectively. (ZIP)peer-reviewedBackground Early life stress is a risk factor for many psychiatric disorders ranging from depression to anxiety. Stress, especially during early life, can induce dysbiosis in the gut microbiota, the key modulators of the bidirectional signalling pathways in the gut-brain axis that underline several neurodevelopmental and psychiatric disorders. Despite their critical role in the development and function of the central nervous system, the effect of n-3 polyunsaturated fatty acids (n-3 PUFAs) on the regulation of gut-microbiota in early-life stress has not been explored. Methods and Results Here, we show that long-term supplementation of eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) (80% EPA, 20% DHA) n-3 PUFAs mixture could restore the disturbed gut-microbiota composition of maternally separated (MS) female rats. Sprague-Dawley female rats were subjected to an early-life stress, maternal separation procedure from postnatal days 2 to 12. Non-separated (NS) and MS rats were administered saline, EPA/DHA 0.4 g/kg/day or EPA/DHA 1 g/kg/day, respectively. Analysis of the gut microbiota in adult rats revealed that EPA/DHA changes composition in the MS, and to a lesser extent the NS rats, and was associated with attenuation of the corticosterone response to acute stress. Conclusions In conclusion, EPA/DHA intervention alters the gut microbiota composition of both neurodevelopmentally normal and early-life stressed animals. This study offers insights into the interaction between n-3 PUFAs and gut microbes, which may play an important role in advancing our understanding of disorders of mood and cognitive functioning, such as anxiety and depression.Research was funded by Food Institutional Research Measure (FIRM) under Grant No. 10/RD/TMFRC/709, the APC Microbiome Institute under Grant No. 07/CE/B1368 and 12/RC/2273, Science Foundation Ireland (SFI) under Grant No. 12/IA/1537

Influential factors in nectar composition and yield in Leptospermum scoparium : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Plant Science, Institute of Agriculture and the Environment, College of Sciences, Massey University, Palmerston North, New Zealand

Material omitted from digital version of thesis: Nickless, E. M., Anderson, C. W. N., Hamilton, G., Stephens, J. M., & Wargent, J. (2016). Soil influences on plant growth, floral density and nectar yield in three cultivars of manuka (Leptospermum scoparium). New Zealand Journal of Botany, 55(2), 100-117. doi:10.1080/0028825X.2016.1247732 ; Nickless, E. M., Holroyd, S. E., Stephens, J. M., Gordon, K. C., & Wargent, J. J. (2014). Analytical FT-Raman spectroscopy to chemotype Leptospermum scoparium and generate predictive models for screening for dihydroxyacetone levels in floral nectar. Journal of Raman Spectroscopy, 45(10), 890-894. doi:10.1002/jrs.4576 ; Nickless, E. M., Holroyd, S. E., Hamilton, G., Gordon, K. C., & Wargent, J. J. (2016). Analytical method development using FTIR-ATR and FT-Raman spectroscopy to assay fructose, sucrose, glucose and dihydroxyacetone, in Leptospermum scoparium nectar. Vibrational Spectroscopy, 84 (2016), 38-43. doi:10.1016/j.vibspec.2016.02.011Leptospermum scoparium (Mānuka) is the plant nectar source for medically bioactive honey, commercially marketed in New Zealand as Unique Mānuka Factor honey (UMF-honey). Methylglyoxal (MGO) is the unique bioactive component of UMF honey with Mānuka nectar containing significant amounts of the carbohydrate dihydroxyacetone (DHA), the chemical precursor for MGO. Anecdotal evidence and recently published data from nectar samples collected from various cultivars in natural sites or botanical gardens has indicated that the DHA and overall composition of L. scoparium nectar varies according to cultivar. The source of this variation is not clearly understood and although there is considerable literature on climatic and genetic influences on nectar composition and yield within various other plant species, there is little published work available on the influence of genetic and environmental factors on the composition and yield of nectar in L. scoparium. Of value to the commercial UMF honey industry in New Zealand is the ability to assess cultivars from breeding programs for the best potential to increase overall UMF honey yield. Predictive modelling of yields is invaluable to the developing honey industry to allow assessment of environmental influences that may affect overall yield along with seasonal influences on nectar production in Mānuka. The research in this thesis establishes the effect of various parameters on overall DHA yield from Mānuka and the beginnings of modelling influencing environmental factors. To determine influences on dihydroxyacetone (DHA) concentration and yield in the nectar of L. scoparium a number of studies were carried out. Methodologies for the collection and analysis of nectar were established. Ten different cultivars of L. scoparium with a range of genetic parentage were studied in controlled glasshouse conditions to assess phenotypic variability in terms of nectar composition and yield as well as plant growth and flowering amongst these cultivars. Significant differences in plant growth and flowering habits were observed amongst the ten cultivars, significant differences in nectar yield and nectar composition with regard to DHA yield were also observed. DHA yields ranged from 2714-7459 mg of DHA/kilogram normalised to 80 oBRIX, with total nectar sugar yields ranging between 0.7 and 4.8 mg amongst the ten cultivars studied. Preliminary research into the effect of temperature, radiation and humidity on nectar composition and yield were also undertaken. Effects of soil composition on these same parameters were researched with a subset of three of the ten cultivars grown on ten different soil types. Plant relative growth rates, dry weights and total plant height were measured throughout a 15 month glasshouse trial. Plant growth, flowering phenology, floral density, nectar yield and DHA composition data was gathered. Soils were analysed for various macronutrient and micronutrient levels and these parameters were modelled against plant data to determine which soil components were influencing plant parameters of interest. Soil type was shown to have no significant effect on DHA concentrations in nectar but results did show that soil type had a significant effect on flowering density amongst the three L. scoparium cultivars studied in the trial. Results from regression analysis of soil chemistry against measured plant parameters indicate that a fertiliser regime has the potential to increase nectar yields due to increased flower numbers. Multivariate analysis using partial least squares regression of soil composition data against plant parameters of value showed that soil components; phosphorus, sulphate, ferric and chloride were commonly shown to influence plant parameters measured. Analytical spectroscopy was investigated as a method to chemotype L. scoparium cultivars and also as a method for quantifying nectar components sucrose, glucose, fructose and DHA. Nectar composition was analysed using high pressure liquid chromatography (HPLC) and compared with fourier transform Raman spectroscopy (FT-Raman) and attenuated total reflectance infrared spectroscopy (ATR-FTIR) analytical spectroscopy methods. FT-Raman spectroscopy was shown to be useful in chemotyping cultivars and in addition proved to be a useful analytical method to predict DHA yield using leaf material from L. scoparium plants from the ten cultivars. FT-Raman and ATR-FTIR proved to be relatively accurate techniques to quantify L. scoparium nectar components DHA, fructose, glucose and sucrose, compared with HPLC methods which use extensive preparation techniques. R-squared values were very good for all nectar components measured excepting the sucrose model at R2 = 0.77. The R2 for the FT-Raman predictions of DHA against HPLC data are very good at 0.85. FTIR prediction data against HPLC data was also good at 0.86 R2. Overall an accurate model is possible for quantifying DHA concentrations in nectar using both FTIR-ATR and FT-Raman spectroscopy. Overall results show that various factors need to be considered when assessing plants for commercial use in the (UMF) Mānuka honey industry within New Zealand. Due to their large impact on overall nectar yield; floral density and plant growth rate parameters are the two key factors of value for commercial assessment of Mānuka cultivars. This research also highlights the importance of assessing not just DHA concentration in deducing cultivar value, but overall nectar yield. These key features must be explored when assessing L. scoparium plants within breeding programs, prior to selection for large-scale field production of high UMF Mānuka honey