Contribution of n-3 Long-Chain Polyunsaturated Fatty Acids to the Prevention of Breast Cancer Risk Factors.

Nowadays, diet and breast cancer are studied at different levels, particularly in tumor prevention and progression. Thus, the molecular mechanisms leading to better knowledge are deciphered with a higher precision. Among the molecules implicated in a preventive and anti-progressive way, n-3 long chain polyunsaturated fatty acids (n-3 LC-PUFAs) are good candidates. These molecules, like docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, are generally found in marine material, such as fat fishes or microalgae. EPA and DHA act as anti-proliferative, anti-invasive, and anti-angiogenic molecules in breast cancer cell lines, as well as in in vivo studies. A better characterization of the cellular and molecular pathways involving the action of these fatty acids is essential to have a realistic image of the therapeutic avenues envisaged behind their use. This need is reinforced by the increase in the number of clinical trials involving more and more n-3 LC-PUFAs, and this, in various pathologies ranging from obesity to a multitude of cancers. The objective of this review is, therefore, to highlight the new elements showing the preventive and beneficial effects of n-3 LC-PUFAs against the development and progression of breast cancer.

Application of pulsed electric fields for the biocompatible extraction of proteins from the microalga Haematococcus pluvialis.

This study aims to employ a pulsed electric field (PEF) treatment for the biocompatible (non-destructive) extraction of proteins from living cells of the green microalga Haematococcus pluvialis. Using a field strength of 1 kV cm-1, we achieved the extraction of 10.2 µg protein per mL of culture, which corresponded to 46% of the total amount of proteins that could be extracted by complete destructive extraction (i.e. the grinding of biomass with glass beads). We found that the extraction yield was not improved by stronger field strengths and was not dependent on the pulse frequency. A biocompatibility index (BI) was defined as the relative abundance of cells that remained alive after the PEF treatment. This index relied on measurements of several physiological parameters after a PEF treatment. It was found that at 1 kV cm-1 that cultures recovered after 72 h. Therefore, these PEF conditions constituted a good compromise between protein extraction efficiency and culture survival. To characterize the PEF treatment further at a molecular level, mass spectrometry-based proteomics analyses of PEF-prepared extracts was used. This led to the identification of 52 electro-extracted proteins. Of these, only 16 proteins were identified when proteins were extracted with PEF at 0.5 cm-1. They belong to core metabolism, stress response and cell movement. Unassigned proteins were also extracted. Their physiological implications and possible utilization in food as alimentary complements are discussed.

Effect of Carotenoids from Phaeodactylum tricornutum on Palmitate-Treated HepG2 Cells.

Non-alcoholic fatty liver disease represents the most common liver disease and is characterized by an excess of lipid accumulation in hepatocytes, mainly stored as triglycerides. Phaeodactylum tricornutum is a marine microalga, which is rich in bioactive molecules known to be hepatoprotective, such as n-3 long-chain polyunsaturated fatty acids and fucoxanthin. The aim of this study was to investigate the effects of a carotenoid extract from P. tricornutum in a cellular model of non-alcoholic fatty liver disease induced by palmitate treatment. The combined effects of carotenoids and lipids, especially n-3 long-chain polyunsaturated fatty acids, were also investigated by using a total lipophilic extract. HepG2 cells were exposed for 24 h to 250 µM palmitate with or without the addition of carotenoid extract (6 µg/mL) or total lipophilic extract (100 µg/mL). The addition of carotenoid extract or total lipophilic extract prevented the accumulation of triglycerides, total cholesterol and cholesterol esters. The carotenoid extract and total lipophilic extract also decreased the mRNA expression levels of genes involved in lipogenesis (ACACA, FASN, SCD and DGAT1) and cholesterol esterification (ACAT1/SOAT1). In addition, the total lipophilic extract also downregulated the LXR/NR1H3 and SREBF1 genes, which are involved in lipogenesis regulation. By contrast, the carotenoid extract increased the mRNA level of CPT1A, a ß-oxidation related gene, and reduced the lipid droplet accumulation. In conclusion, this study highlights the preventive effects against non-alcoholic fatty liver disease of the two microalga extracts.

Transcriptomic Response of Breast Cancer Cells MDA-MB-231 to Docosahexaenoic Acid: Downregulation of Lipid and Cholesterol Metabolism Genes and Upregulation of Genes of the Pro-Apoptotic ER-Stress Pathway.

Despite considerable efforts in prevention and therapy, breast cancer remains a major public health concern worldwide. Numerous studies using breast cancer cell lines have shown the antiproliferative and pro-apoptotic effects of docosahexaenoic acid (DHA). Some studies have also demonstrated the inhibitory effect of DHA on the migration and invasion of breast cancer cells, making DHA a potential anti-metastatic agent. Thus, DHA has shown its potential as a chemotherapeutic adjuvant. However, the molecular mechanisms triggering DHA effects remain unclear, and the aim of this study was to provide a transcriptomic basis for further cellular and molecular investigations. Therefore, MDA-MB-231 cells were treated with 100 µM DHA for 1`2 h or 24 h before RNA-seq analysis. The results show the great impact of DHA-treatment on the transcriptome, especially after 24 h of treatment. The impact of DHA is particularly visible in genes involved in the cholesterol biosynthesis pathway that is strongly downregulated, and the endoplasmic reticulum (ER)-stress response that is, conversely, upregulated. This ER-stress and unfolded protein response could explain the pro-apoptotic effect of DHA. The expression of genes related to migration and invasion (especially SERPINE1, PLAT, and MMP11) is also impacted by DHA. In conclusion, this transcriptomic analysis supports the antiproliferative, pro-apoptotic and anti-invasive effects of DHA, and provides new avenues for understanding its molecular mechanisms.

Microalgal Fatty Acids and Their Implication in Health and Disease.

BACKGROUND: The fatty acids of seed plants and microalgae stored in triglyceride are all produced in the plastid and incorporated into triglycerides by a complex biochemical exchange between the plastid envelope and the endoplasmic reticulum. The oils of seed plants provide the basis for vegetal fat production and the microalgal fats represent an important part of the basal food web of the marine environment. The health-promoting properties of these various sources of fats and in particular the long-chain polyunsaturated fatty acids of marine microalgae are widely recognized. The omega-3 fatty acids are known to have benefits on health and disease. Indeed, alpha-linolenic, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) are linked to the regulation of mechanisms involved in numerous biological functions associated with cardiovascular disease and cancer prevention. Most EPA and DHA sources for human nutrition are provided by decreasing global stocks of fish. This is one of the reasons why industrial research has been directed towards more sustainable sources of these "marine" lipids. The synthesis of fatty acids and triglycerides are in many respects similar in higher plants and marine algae, but there are also important differences. CONCLUSION: This mini-review covers the biochemistry of fatty acid and lipid synthesis in marine microalgae, and the potential health impact of the different fats is also discussed.

Diatom milking: a review and new approaches.

The rise of human populations and the growth of cities contribute to the depletion of natural resources, increase their cost, and create potential climatic changes. To overcome difficulties in supplying populations and reducing the resource cost, a search for alternative pharmaceutical, nanotechnology, and energy sources has begun. Among the alternative sources, microalgae are the most promising because they use carbon dioxide (CO2) to produce biomass and/or valuable compounds. Once produced, the biomass is ordinarily harvested and processed (downstream program). Drying, grinding, and extraction steps are destructive to the microalgal biomass that then needs to be renewed. The extraction and purification processes generate organic wastes and require substantial energy inputs. Altogether, it is urgent to develop alternative downstream processes. Among the possibilities, milking invokes the concept that the extraction should not kill the algal cells. Therefore, it does not require growing the algae anew. In this review, we discuss research on milking of diatoms. The main themes are (a) development of alternative methods to extract and harvest high added value compounds; (b) design of photobioreactors;

Docosahexaenoic acid inhibits the invasion of MDA-MB-231 breast cancer cells through upregulation of cytokeratin-1.

Docosahexaenoic acid (DHA), the main member of the omega-3 essential fatty acid family, has been shown to reduce the invasion of the triple-negative breast cancer cell line MDA-MB-231, but the mechanism involved remains unclear. In the present study, a proteomic approach was used to define changes in protein expression induced by DHA. Proteins from crude membrane preparations of MDA-MB-231 cells treated with 100 µM DHA were separated by two-dimensional electrophoresis (2-DE) and differentially expressed proteins were identified using MALDI-TOF mass spectrometry. The main changes observed were the upregulation of Keratin, type Ⅱ cytoskeletal 1 (KRT1), catalase and lamin-A/C. Immunocytochemistry analyses confirmed the increase in KRT1 induced by DHA. Furthermore, in vitro invasion assays showed that siRNA against KRT1 was able to reverse the DHA-induced inhibition of breast cancer cell invasion. In conclusion, KRT1 is involved in the anti-invasive activity of DHA in breast cancer cells.

E. coli electroeradication on a closed loop circuit by using milli-, micro- and nanosecond pulsed electric fields: comparison between energy costs.

One of the different ways to eradicate microorganisms, and particularly bacteria that might have an impact on health consists in the delivery of pulsed electric fields (PEFs). The technologies of millisecond (ms) or microsecond (µs) PEF are still well known and used for instance in the process of fruit juice sterilization. However, this concept is costly in terms of delivered energy which might be too expensive for some other industrial processes. Nanosecond pulsed electric fields (nsPEFs) might be an alternative at least for lower energetic cost. However, only few insights were available and stipulate a gain in cost and in efficiency as well. Using Escherichia coli, the impact of frequency and low rate on eradication and energy consumption by msPEF, µsPEF and nsPEF have been studied and compared. While a 1 log10 was reached with an energy cost of 100 and 158 kJ/L with micro- and millisecond PEFs respectively, nsPEF reached the reduction for similar energy consumption. The best condition was obtained for a 1 log10 deactivation in 0.5h, for energy consumption of 143 kJ/L corresponding to 0.04 W · h when the field was around 100 kV/cm. Improvement can also be expected by producing a generator capable to increase the electric field.

Optimization of protein electroextraction from microalgae by a flow process.

Classical methods, used for large scale treatments such as mechanical or chemical extractions, affect the integrity of extracted cytosolic protein by releasing proteases contained in vacuoles. Our previous experiments on flow processes electroextraction on yeasts proved that pulsed electric field technology allows preserving the integrity of released cytosolic proteins, by not affecting vacuole membranes. Furthermore, large cell culture volumes are easily treated by the flow technology. Based on this previous knowledge, we developed a new protocol in order to electro-extract total cytoplasmic proteins from microalgae (Nannochloropsis salina, Chlorella vulgaris and Haematococcus pluvialis). Given that induction of electropermeabilization is under the control of target cell size, as the mean diameter for N. salina is only 2.5 µm, we used repetitive 2 ms long pulses of alternating polarities with stronger field strengths than previously described for yeasts. The electric treatment was followed by a 24h incubation period in a salty buffer. The amount of total protein release was observed by a classical Bradford assay. A more accurate evaluation of protein release was obtained by SDS-PAGE. Similar results were obtained with C. vulgaris and H. pluvialis under milder electrical conditions as expected from their larger size.

Inactivation of Bacillus subtilis var. niger of both spore and vegetative forms by means of corona discharges applied in water.

Spores are dormant units of bacteria resistant to numerous disinfection methods. Additionally, the effects on bacteria of repetitive electrical discharges in water by used of the so-called "corona discharges" or streamer are poorly described. In this study vegetative and spore forms of Bacillus subtilis var. niger were subjected to these discharges. To generate corona discharges in water, a Marx generator capable of delivering 60-90 kV was used with a coaxial chamber of treatment. Vegetative and spore form reductions were defined using colony-forming unit counting. Proteins extracts were separated by two-dimensional electrophoresis and spots of interest were characterized by mass spectrometry. Shock waves were assessed by the diminution of liposome size and OD(400 nm). The results show a decrease in bacteria viability of 2 log(10) after 1000 discharges on the vegetative form and 4 log(10) after 10,000 discharges on the spores. Two-dimensional electrophoresis showed that the streamers impact the regulation of several proteins in the vegetative forms with UniProt ID: P80861, Q06797, P80244, C0ZI91, respectively. The reduction appears to be due, in part, to hydrogen peroxide (H(2)O(2)) generated by the corona discharges while spore deactivation remained insensitive to these chemicals. The spore eradication was associated to shock waves induced by the discharges but not H(2)O(2). Corona discharges appear as a prospective method for eradication of spores in water. The corona discharges can be an efficient method for decontamination processes of waste water.

The janus face of lipids in human breast cancer: how polyunsaturated Fatty acids affect tumor cell hallmarks.

For several years, lipids and especially n - 3 and n - 6 polyunsaturated fatty acids (PUFAs) receive much attention in human health. Epidemiological studies tend to correlate a PUFA-rich diet with a reduced incidence of cancer, including breast cancer. However, the molecular and cellular mechanisms supporting the effect of PUFAs in breast cancer cells remain relatively unknown. Here, we review some recent progress in understanding the impact that PUFA may have on breast cancer cell proliferation, apoptosis, migration, and invasion. While most of the results obtained with docosahexaenoic acid and/or eicosapentaenoic acid show a decrease of tumor cell proliferation and/or aggressivity, there is some evidence that other lipids, which accumulate in breast cancer tissues, such as arachidonic acid may have opposite effects. Finally, lipids and especially PUFAs appear as potential adjuvants to conventional cancer therapy.

Proteome changes induced by overexpression of the p75 neurotrophin receptor (p75NTR) in breast cancer cells.

In breast cancer cells, the neurotrophin receptor p75(NTR) acts as a prosurvival factor able to stimulate resistance to apoptosis, but its mechanism of action remains incompletely defined. In this study, we investigated the global proteome modification induced by p75(NTR) overexpression in breast cancer cells treated by the pro-apoptotic agent tumor necrosis factor (TNF)-related-apoptosis-inducing-ligand (TRAIL). p75(NTR) was stably overexpressed in the MCF-7 breast cancer cells and the impact of a treatment by TRAIL was investigated in wild type vs. p75(NTR) overexpressing cells. Proteins were separated in two-dimensional electrophoresis, and regulated spots were detected by computer assisted analysis before identification by MALDI-TOF/TOF mass spectrometry. In the absence of TRAIL treatment, p75(NTR) did not induce any change in the proteome of breast cancer cells. In contrast, after treatment with TRAIL, fragments of cytokeratin-8, -18 and -19, as well as full length cytokeratin-18, were up-regulated by p75(NTR) overexpression. Of note, spectrin alpha-chain and the ribosomal protein RPLP0 were induced by TRAIL, independently of p75(NTR) level. Interestingly, the well known stress-induced protein HSP-27 was less abundant when p75(NTR) was overexpressed, indicating that p75(NTR) overexpression reduced TRAIL induced cell stress. These data indicate that overexpression of p75(NTR) induces proteome modifications in breast cancer cells and provide information on how this receptor contributes in tumor cell resistance to apoptosis.

Docosahexaenoic acid intake decreases proliferation, increases apoptosis and decreases the invasive potential of the human breast carcinoma cell line MDA-MB-231.

Breast cancer is the most common cancer in women in industrialized countries. Environmental factors, such as differences in diet are likely to have an important influence on cancer emergence. Among these factors, n-3 polyunsaturated-fatty acids, such as docosahexaenoic acid (DHA), are good candidates for preventing breast cancer. Here we investigate the effect of DHA on the human breast cancer cell line MDA-MB-231 and show that DHA incorporation i) has an anti-proliferative effect, ii) induces apoptosis via a transient increase in caspase-3 activity and the promotion of nuclear condensation, and iii) reduces the invasive potential of MDA-MB-231 cells. To conclude, DHA may have beneficial effects as a result of slowing the proliferation of tumor cells, and minimizing their metastatic potential.

Partial characterization of endothelial FGF receptor functional domain by monoclonal antibody VBS-1.

Polypeptide growth factors mediate their cellular responses by binding to and activating specific cell surface receptors. Monoclonal antibody (MAb) VBS-1, produced against native fibroblast growth factor receptor-1 (FGFR-1), inhibited the binding of fibroblast growth factor-2 (FGF-2) to its receptor on coronary venular endothelial cells (CVECs) as determined by 125I-FGF-2 Scatchard analysis and [3H]thymidine uptake assays (ED50 = 80 ng/mL). Enzyme studies demonstrated that MAb VBS-1 binds to a protein epitope. Proteolytic mapping of the CVEC-FGFR established that a 52 kDa doublet contained the FGF binding site and the MAb VBS-1 antigenic epitope. N-glycanase digestion suggested the presence of a 50 kDa core protein for the CVEC-FGFR. Tunicamycin treatment resulted in the loss of expression of the core protein and the mature receptor, indicating the importance of CVEC-FGFR n-linked glycosylation. By Northern blot analysis, it was determined that CVECs express fgfr-1 and not fgfr-2. VBS-1 recognized FGFR-1 (140 kDa) and crossreacted weakly with FGFR-2 (135 kDa). Using a combination of affinity crosslinking, proteolytic mapping and Mab VBS-1 binding studies, we have located the FGF binding site near the NH2-terminal domain of the receptor close to the highly acidic box.