Omega-3 fatty acid treatment combined with chemotherapy to prevent toxicity, drug resistance, and metastasis in cancer

: Despite advances in treatment, individuals diagnosed with cancer are often at risk of suffering from metastasis, tumor recurrence, therapy resistance, and off-target toxicities from conventional chemo-, radio-, and endocrine- therapies. Drugs with potent anticancer and antimetastatic activity, but with milder side effects, can be combined with conventional therapies to increase efficacy, reduced therapy resistance, and decrease toxicity. Substantial data from epidemiological, cell culture, animal and clinical studies have established anticancer potential of nontoxic omega-3 fatty acids. This paper highlights the beneficial effects of omega-3 fatty acid treatment when used in combination with conventional therapies protect against metastasis, enhance therapeutic efficacy, and prevent the off-target toxicity caused by conventional therapies. These omega-3 fatty acids target therapy-induced central players NF-κB, and ROS to prevent drug-associated metastasis, therapy resistance, and off-target toxicities.

P–208 Oleic acid rescues altered autophagy induced by palmitic acid during mouse preimplantation development

Study question The aim of the study is to identify the autophagic profile and the effects of fatty acid treatments on autophagic activity in preimplantation mouse embryos. Summary answer Autophagic activity varies significantly in early stages of mouse preimplantation development; exposure to fatty acids alters the embryonic autophagy profile. What is known already Obesity is one of the top comorbidities for infertility, and obese individuals have elevated fatty acid levels. In serum, palmitic acid (PA) and oleic acid (OA) are the most abundant saturated and unsaturated fatty acids, respectively. We recently reported that PA impairs blastocyst development, affects mitochondrial reactive oxygen species, triacylglycerol levels, and endoplasmic reticulum stress pathways during mouse preimplantation development. Interestingly, the addition of OA counteracts those effects. Autophagy plays an essential role in embryo development, as knock-out of a key autophagy protein is embryonic lethal. Little is known about the autophagic profile in fatty acid treated mouse preimplantation embryos. Study design, size, duration Pools of 20 – 25 mouse embryos were collected from gonadotrophin super-ovulated and mated CD1 female mice. Two-cell stage embryos were treated with 100 µM PA and 250 µM OA, alone and in combination, and 1.5% bovine serum albumin media (control) within KSOMaa media for 18, 24, and 48 hours in vitro. The detection of various autophagic markers were evaluated by immunofluorescence microscopy and RT-qPCR. Participants/materials, setting, methods mRNA levels of autophagic markers were measured using RT-qPCR with the Taqman primers and Universal PCR Mix. Immunofluorescence staining of LC3 puncta (marker for autophagosome formation) was performed using LC3A/B polyclonal antibody (Invitrogen PA1–16931) and DAPI (4′,6-Diamidino–2-phenylindole dihydrochloride) was used to stain for cell nuclei. Analysis of LC3 puncta was performed using ImageJ software. Images were acquired using an LSM 800 laser scanning confocal microscope. Data analysis was completed by GraphPad Prism software. Main results and the role of chance Mouse preimplantation embryos showed no change in mRNA levels of autophagic markers (Bcln1, ATG3, ATG5, and LC3) relative to the control group after 48-hours exposure of 100 µM PA and 250 µM OA treatments, alone and in combination. The number of LC3 puncta was measured and analyzed as a reflection of autophagic activity in mouse preimplantation embryos. Under the fatty acid-free condition, the average number of LC3 puncta per blastomere was significantly decreased after 18 hours of development (p < 0.005). However, the average number of LC3 puncta per blastomere at 18, 24, and 48 hours were not significantly different from each other (p = 0.2724). Following 100 µM PA and 250 µM OA treatments, alone and in combination, autophagic activity was impacted by the presence of fatty acids. Mouse preimplantation embryos exposed to control and fatty acid treatment groups demonstrated no significant differences in LC3 puncta per blastomere at 18- and 24-hours treatment time (p = 0.5381; p = 0.7829). However, embryos exposed to 48 hours of PA treatment had a significantly greater number of LC3 puncta per blastomere than embryos exposed to 48 hours of OA and PA and OA combination treatments (p < 0.05). Limitations, reasons for caution Although LC3 puncta count (autophagosome formation) is impacted by fatty acid treatment, autophagic flux must be measured to fully investigate autophagic activity during mouse preimplantation development. These processes need to be measured in human embryos cultured in vitro. Wider implications of the findings: Profiling autophagic activity in fatty acid treated mouse preimplantation embryos would guide future investigations on pharmacological modulation of autophagy as a therapeutic intervention for developmentally delayed embryos. With the information gained, we aim to develop strategies to assist overweight and obese patients with their fertility needs. Trial registration number Not applicable

Combined prenatal Lactobacillus reuteri and ω-3 supplementation synergistically modulates DNA methylation in neonatal T helper cells

Background Environmental exposures may alter DNA methylation patterns of T helper cells. As T helper cells are instrumental for allergy development, changes in methylation patterns may constitute a mechanism of action for allergy preventive interventions. While epigenetic effects of separate perinatal probiotic or ω-3 fatty acid supplementation have been studied previously, the combined treatment has not been assessed. We aimed to investigate epigenome-wide DNA methylation patterns from a sub-group of children in an on-going randomised double-blind placebo-controlled allergy prevention trial using pre- and postnatal combined Lactobacillus reuteri and ω-3 fatty acid treatment. To this end, > 866000 CpG sites (MethylationEPIC 850K array) in cord blood CD4+ T cells were examined in samples from all four study arms (double-treatment: n = 18, single treatments: probiotics n = 16, ω-3 n = 15, and double placebo: n = 14). Statistical and bioinformatic analyses identified treatment-associated differentially methylated CpGs and genes, which were used to identify putatively treatment-induced network modules. Pathway analyses inferred biological relevance, and comparisons were made to an independent allergy data set. Results Comparing the active treatments to the double placebo group, most differentially methylated CpGs and genes were hypermethylated, possibly suggesting induction of transcriptional inhibition. The double-treated group showed the largest number of differentially methylated CpGs, of which many were unique, suggesting synergy between interventions. Clusters within the double-treated network module consisted of immune-related pathways, including T cell receptor signalling, and antigen processing and presentation, with similar pathways revealed for the single-treatment modules. CpGs derived from differential methylation and network module analyses were enriched in an independent allergy data set, particularly in the double-treatment group, proposing treatment-induced DNA methylation changes as relevant for allergy development. Conclusion Prenatal L. reuteri and/or ω-3 fatty acid treatment results in hypermethylation and affects immune- and allergy-related pathways in neonatal T helper cells, with potentially synergistic effects between the interventions and relevance for allergic disease. Further studies need to address these findings on a transcriptional level, and whether the results associate to allergy development in the children. Understanding the role of DNA methylation in regulating effects of perinatal probiotic and ω-3 interventions may provide essential knowledge in the development of efficacious allergy preventive strategies. Trial registration ClinicalTrials.gov, ClinicalTrials.gov-ID: NCT01542970. Registered 27th of February 2012—Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT01542970.

Combined prenatal Lactobacillus reuteri and ω-3 supplementation synergistically modulates DNA methylation in neonatal T helper cells

ABSTRACTBackgroundEnvironmental exposures may alter DNA methylation patterns of T helper cells. As T helper cells are instrumental for allergy development, changes in methylation patterns may constitute a mechanism of action for allergy preventive interventions. While epigenetic effects of separate perinatal probiotic or ω-3 fatty acid supplementation have been studied previously, the combined treatment has not been assessed. We aimed to investigate epigenome-wide DNA methylation patterns in cord blood samples from children in a randomised double-blind placebo-controlled allergy prevention trial using pre- and postnatal combined Lactobacillus reuteri and ω-3 fatty acid treatment. To this end, >866 000 CpG sites (MethylationEPIC 850K array) in cord blood CD4+ T cells were examined in samples from all four study arms (double-treatment: n=18, single treatments: probiotics n=16, ω-3 n=15, and double placebo: n=14). Statistical and bioinformatic analyses identified treatment-associated differentially methylated CpGs and genes, which were used to identify treatment-induced network modules. Pathway analyses inferred biological relevance, and comparisons were made to an independent allergy data set.ResultsComparing the active treatments to the double placebo group, most differentially methylated CpGs and genes were hypermethylated, suggesting induction of transcriptional inhibition. The double-treated group showed the largest number of differentially methylated CpGs, of which many were unique, suggesting synergy between interventions. Clusters within the double-treated network module consisted of immune-related pathways, including T cell receptor signalling, and antigen processing and presentation, with similar pathways revealed for the single-treatment modules. CpGs derived from differential methylation and network module analyses were enriched in an independent allergy data set, particularly in the double-treatment group, proposing treatment-induced DNA methylation changes as relevant for allergy development.ConclusionPrenatal L. reuteri and/or ω-3 fatty acid treatment results in hypermethylation and affects immune- and allergy-related pathways in neonatal T helper cells, with potentiallysynergistic effects between the interventions and relevance for allergic disease. Further studies need to address these findings on a transcriptional level, and whether the results associate to allergy development in the children. Understanding the role of DNA methylation in regulating effects of perinatal probiotic and ω-3 interventions may provide essential knowledge in the development of efficacious allergy preventive strategies.Trial registrationClinicalTrials.gov, ClinicalTrials.gov-ID: NCT01542970. Registered 27th of February 2012 – Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT01542970

Effect of Carbon Nanostructures and Fatty Acid Treatment on the Mechanical and Thermal Performances of Flax/Polypropylene Composites

Four different strategies for mitigating the highly hydrophilic nature of flax fibers were investigated with a view to increase their compatibility with apolar polypropylene. The effects of two carbon nanostructures (graphene nanoplatelets (GNPs) and carbon nanotubes (CNTs)), of a chemical modification with a fatty acid (stearic acid), and of maleated polypropylene on interfacial adhesion, mechanical properties (tensile and flexural), and thermal stability (TGA) were compared. The best performance was achieved by a synergistic combination of GNPs and maleated polypropylene, which resulted in an increase in tensile strength and modulus of 42.46% and 54.96%, respectively, compared to baseline composites. Stearation proved to be an effective strategy for increasing the compatibility with apolar matrices when performed in an ethanol solution with a 0.4 M concentration. The results demonstrate that an adequate selection of surface modification strategies leads to considerable enhancements in targeted properties.

Differential Effects of Lipid and Vitamin D treatment on Cell Viability and CYP24A1 Expression in Hepatocytes and Hepatic Stellate Cells

Activated hepatic stellate cells (HSCs) are a key contributor to liver fibrosis and drive the progression to advanced disease for many liver conditions, including non-alcoholic fatty liver disease. Previous studies suggest vitamin D may reduce inflammatory and pro-fibrogenic activity of HSCs in vitro. However, the mechanisms underpinning the effects of vitamin D in HSCs are not fully understood. The overall aim of these experiments was to mimic a lipid loading model on immortalised HSCs to test their responses to 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). Two different human immortalised cell lines: HepG2, hepatocellular carcinoma cells, and LX-2, hepatic stellate cells; were cultured using standard methods. Cell viability in different treatment vehicles (2% DMSO and/or 0.1% ethanol) under serum free conditions was measured by MTT assay after 6 and 24 h. Cells were cultured with increasing concentrations of fatty acids (0–500μM, 1:1 oleic acid: palmitic acid) or vitamin D. Nile red, a neutral lipophilic fluorescent dye, was used to measure total intracellular lipid and quantified relative to vehicle. CYP24A1 mRNA expression was measured by qPCR in response to 1000nM 1α,25(OH)2D3 treatment in both cell lines for 24 h using TaqMan® gene expression assays and normalised to 18S rRNA. Cell viability in response to vehicle was examined at 6 h and 24 h to determine the optimal experimental time points. Whereas, HepG2 cells remained unaffected at 24 h in response to either or both vehicles combined (n = 4; combined vehicles, P = 0.3187), LX-2 cells showed reduced viability even at 6 h (n = 5; combined vehicles, P = 0.0050). Fatty acid treatment led to intracellular lipid accumulation in both cell lines. In response to 500μM fatty acid treatment, intracellular lipid increased by 1.7-fold in LX-2 cells at 6 h (n = 5, P = 0.00174) and 3.9-fold in HepG2 cells after 24 h (n = 4, P = 0.00184). Notably, CYP24A1 mRNA expression was markedly induced by vitamin D treatment in LX-2 cells (136 ± 7.64-fold, n = 3, P = 0.0010) in comparison to HepG2 cells (22 ± 0.78-fold, n = 3, P < 0.0001). In summary, the cell viability data suggested optimal time points for both fatty acid and vitamin D treatments may be 6 h for LX-2 cells, and 24 h for HepG2 cells. While intracellular lipid accumulation differed between the cell lines in response to fatty acid treatment, both cell lines produced a dose-dependent increase in intracellular lipid. Lastly, CYP24A1 mRNA expression confirmed the responsiveness of both cell types to vitamin D treatment. Ongoing experiments are examining microRNA expression in HSCs in response to both vitamin D and lipid loading.