Paola Antonia Corsetto

Effects of n-3 PUFAs on breast cancer cells through their incorporation in plasma membrane.

BackgroundPUFAs are important molecules for membrane order and function; they can modify inflammation-inducible cytokines production, eicosanoid production, plasma triacylglycerol synthesis and gene expression. Recent studies suggest that n-3 PUFAs can be cancer chemopreventive, chemosuppressive and auxiliary agents for cancer therapy. N-3 PUFAs could alter cancer growth influencing cell replication, cell cycle, and cell death. The question that remains to be answered is how n-3 PUFAs can affect so many physiological processes. We hypothesize that n-3 PUFAs alter membrane stability, modifying cellular signalling in breast cancer cells.MethodsTwo lines of human breast cancer cells characterized by different expression of ER and EGFR receptors were treated with AA, EPA or DHA. We have used the MTT viability test and expression of apoptotic markers to evaluate the effect of PUFAs on cancer growth. Phospholipids were analysed by HPLC/GC, to assess n-3 incorporation into the cell membrane.ResultsWe have observed that EPA and DHA induce cell apoptosis, a reduction of cell viability and the expression of Bcl2 and procaspase-8. Moreover, DHA slightly reduces the concentration of EGFR but EPA has no effect. Both EPA and DHA reduce the activation of EGFR.N-3 fatty acids are partially metabolized in both cell lines; AA is integrated without being further metabolized. We have analysed the fatty acid pattern in membrane phospholipids where they are incorporated with different degrees of specificity. N-3 PUFAs influence the n-6 content and vice versa.ConclusionsOur results indicate that n-3 PUFA feeding might induce modifications of breast cancer membrane structure that increases the degree of fatty acid unsaturation. This paper underlines the importance of nutritional factors on health maintenance and on disease prevention.

Endogenous Antioxidants and Radical Scavengers

All living organisms are constantly exposed to oxidant agents deriving from both endogenous and exogenous sources capable to modify biomolecules and induce damages. Free radicals generated by oxidative stress exert an important role in the development of tissue damage and aging. Reactive species (RS) derived from oxygen (ROS) and nitrogen (RNS) pertain to free radicals family and are constituted by various forms of activated oxygen or nitrogen. RS are continuosly produced during normal physiological events but can be removed by antioxidant defence mechanism: the imbalance between RS and antioxidant defence mechanism leads to modifications in cellular membrane or intracellular molecules. In this chapter only endogenous antioxidant molecules will be critically discussed, such as Glutathione, Alpha-lipoic acid, Coenzyme Q, Ferritin, Uric acid, Bilirubin, Metallothioneine, L-carnitine and Melatonin.

Antioxidant defences in hydrated and desiccated states of the tardigrade Paramacrobiotus richtersi

Reactive oxygen species (ROS) are formed in all aerobic organisms, potentially leading to oxidative damage of all biological molecules. A number of defence mechanisms have developed to protect the organism from attack by ROS. Desiccation tolerance is correlated with an increase in the antioxidant potential in several organisms, but the regulation of the antioxidant defence system is complex and its role in desiccation-tolerant organisms is not yet firmly established. To determine if anhydrobiotic tardigrades have an antioxidant defence system, capable of counteracting ROS, we compared the activity of several antioxidant enzymes, the fatty acid composition and Heat shock protein expression in two physiological states (desiccated vs. hydrated) of the tardigrade Paramacrobiotus richtersi. In hydrated tardigrades, superoxide dismutase and catalase show comparable activities, while in desiccated specimens the activity of superoxide dismutase increases. Both glutathione peroxidase and glutathione were induced by desiccation. The percentage of fatty acid composition of polyunsaturated fatty acids and the amount of thiobarbituric acid reactive substances are higher in desiccated animals than in hydrated ones. Lastly, desiccated tardigrades did not differ significantly from the hydrated ones in the relative levels of Hsp70 and Hsp90. These results indicate that the possession of antioxidant metabolism could represent a crucial strategy to avoid damages during desiccation in anhydrobiotic tardigrades.

Omega 3 fatty acids chemosensitize multidrug resistant colon cancer cells by down-regulating cholesterol synthesis and altering detergent resistant membranes composition

BackgroundThe activity of P-glycoprotein (Pgp) and multidrug resistance related protein 1 (MRP1), two membrane transporters involved in multidrug resistance of colon cancer, is increased by high amounts of cholesterol in plasma membrane and detergent resistant membranes (DRMs). It has never been investigated whether omega 3 polyunsatured fatty acids (PUFAs), which modulate cholesterol homeostasis in dyslipidemic syndromes and have chemopreventive effects in colon cancer, may affect the response to chemotherapy in multidrug resistant (MDR) tumors.MethodsWe studied the effect of omega 3 PUFAs docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) in human chemosensitive colon cancer HT29 cells and in their MDR counterpart, HT29-dx cells.ResultsMDR cells, which overexpressed Pgp and MRP1, had a dysregulated cholesterol metabolism, due to the lower expression of ubiquitin E3 ligase Trc8: this produced lower ubiquitination rate of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCoAR), higher cholesterol synthesis, higher cholesterol content in MDR cells. We found that DHA and EPA re-activated Trc8 E3 ligase in MDR cells, restored the ubiquitination rate of HMGCoAR to levels comparable with chemosensitive cells, reduced the cholesterol synthesis and incorporation in DRMs. Omega 3 PUFAs were incorporated in whole lipids as well as in DRMs of MDR cells, and altered the lipid composition of these compartments. They reduced the amount of Pgp and MRP1 contained in DRMs, decreased the transporters activity, restored the antitumor effects of different chemotherapeutic drugs, restored a proper tumor-immune system recognition in response to chemotherapy in MDR cells.ConclusionsOur work describes a new biochemical effect of omega 3 PUFAs, which can be useful to overcome chemoresistance in MDR colon cancer cells.

Effects of Long-Term Space Flight on Erythrocytes and Oxidative Stress of Rodents

Erythrocyte and hemoglobin losses have been frequently observed in humans during space missions; these observations have been designated as “space anemia”. Erythrocytes exposed to microgravity have a modified rheology and undergo hemolysis to a greater extent. Cell membrane composition plays an important role in determining erythrocyte resistance to mechanical stress and it is well known that membrane composition might be influenced by external events, such as hypothermia, hypoxia or gravitational strength variations. Moreover, an altered cell membrane composition, in particular in fatty acids, can cause a greater sensitivity to peroxidative stress, with increase in membrane fragility. Solar radiation or low wavelength electromagnetic radiations (such as gamma rays) from the Earth or the space environment can split water to generate the hydroxyl radical, very reactive at the site of its formation, which can initiate chain reactions leading to lipid peroxidation. These reactive free radicals can react with the non-radical molecules, leading to oxidative damage of lipids, proteins and DNA, etiologically associated with various diseases and morbidities such as cancer, cell degeneration, and inflammation. Indeed, radiation constitutes on of the most important hazard for humans during long-term space flights. With this background, we participated to the MDS tissue-sharing program performing analyses on mice erythrocytes flown on the ISS from August to November 2009. Our results indicate that space flight induced modifications in cell membrane composition and increase of lipid peroxidation products, in mouse erythrocytes. Moreover, antioxidant defenses in the flight erythrocytes were induced, with a significant increase of glutathione content as compared to both vivarium and ground control erythrocytes. Nonetheless, this induction was not sufficient to prevent damages caused by oxidative stress. Future experiments should provide information helpful to reduce the effects of oxidative stress exposure and space anemia, possibly by integrating appropriate dietary elements and natural compounds that could act as antioxidants.

Comparison between the AA/EPA ratio in depressed and non depressed elderly females: omega-3 fatty acid supplementation correlates with improved symptoms but does not change immunological parameters

BackgroundDepression is one of the most frequently missed diagnoses in elderly people, with obvious negative effects on quality of life. Various studies have shown that long chain omega-3 polyunsaturated fatty acids (n-3 PUFA) may be useful in its management. Our objective was to evaluate whether a supplement containing n-3 PUFA improves depressive symptoms in depressed elderly patients, and whether the blood fatty acid pattern is correlated with these changes.MethodsThe severity of depressive symptoms according to the Geriatric Depression Scale (GDS), blood fatty acid composition and erythrocyte phospholipids were analyzed in 46 depressed females aged 66-95y, diagnosed with depression according to DSMIV, within the context of a randomized, double-blind, placebo-controlled trial. 22 depressed females were included in the intervention group (2.5 g/day of n-3 PUFA for 8 weeks), and 24 in the placebo group. We also measured immunological parameters (CD2, CD3, CD4, CD8, CD16, CD19 and cytokines (IL-5, IL-15).ResultsThe mean GDS score and AA/EPA ratio, in whole blood and RBC membrane phospholipids, were significantly lower after 2 months supplementation with n-3 PUFA. A significant correlation between the amelioration of GDS and the AA/EPA ratio with some immunological parameters, such as CD2, CD19, CD4, CD16 and the ratio CD4/CD8, was also found. Nevertheless, omega-3 supplementation did not significantly improve the studied immunological functions.Conclusionsn-3 PUFA supplementation ameliorates symptoms in elderly depression. The n-3 PUFA status may be monitored by means of the determination of whole blood AA/EPA ratio.

Atomic force microscopy imaging of lipid rafts of human breast cancer cells

Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-1.

Repeated Intratracheal Instillation of PM10 Induces Lipid Reshaping in Lung Parenchyma and in Extra-Pulmonary Tissues

Adverse health effects of air pollution attributed mainly to airborne particulate matter have been well documented in the last couple of decades. Short term exposure, referring to a few hours exposure, to high ambient PM10 concentration is linked to increased hospitalization rates for cardiovascular events, typically 24 h after air pollution peaks. Particulate matter exposure is related to pulmonary and cardiovascular diseases, with increased oxidative stress and inflammatory status. Previously, we have demonstrated that repeated intratracheal instillation of PM10sum in BALB/c mice leads to respiratory tract inflammation, creating in lung a condition which could potentially evolve in a systemic toxic reaction. Additionally, plasma membrane and tissue lipids are easily affected by oxidative stress and directly correlated with inflammatory products. With this aim, in the present investigation using the same model, we analyzed the toxic potential of PM10sum exposure on lipid plasma membrane composition, lipid peroxidation and the mechanisms of cells protection in multiple organs such as lung, heart, liver and brain. Obtained results indicated that PM10 exposure led to lung lipid reshaping, in particular phospholipid and cholesterol content increases; concomitantly, the generation of oxidative stress caused lipid peroxidation. In liver we found significant changes in lipid content, mainly due to an increase of phosphatidylcholine, and in total fatty acid composition with a more pronounced level of docosahexaenoic acid; these changes were statistically correlated to lung molecular markers. Heart and brain were similarly affected; heart was significantly enriched in triglycerides in half of the PM10sum treated mice. These results demonstrated a direct involvement of PM10sum in affecting lipid metabolism and oxidative stress in peripheral tissues that might be related to the serious systemic air-pollution effects on human health.

Two ABCB4 point mutations of strategic NBD-motifs do not prevent protein targeting to the plasma membrane but promote MDR3 dysfunction

The ABCB4 gene encodes for MDR3, a protein that translocates phosphatidylcholine from the inner to the outer leaflet of the hepatocanalicular membrane; its deficiency favors the formation of ‘toxic bile’. Several forms of hepatobiliary diseases have been associated with ABCB4 mutations, but the detrimental effects of most mutations on the encoded protein needs to be clarified. Among subjects with cholangiopathies who were screened for mutations in ABCB4 by direct sequencing, we identified the new mutation p.(L481R) in three brothers. According to our model of tertiary structure, this mutation affects the Q-loop, whereas the p.(Y403H) mutation, that we already described in two other families, involves the A-loop. This study was aimed at analyzing the functional relevance of these two ABCB4 mutations: MDR3 expression and lipid content in the culture supernatant were evaluated in cell lines stably transfected with the ABCB4 wild-type clone and corresponding mutants. No differences of expression were observed between wild-type and mutant gene products. Instead, both mutations caused a reduction of phosphatidylcholine secretion compared with the wild-type transfected cell lines. On the contrary, cholesterol (Chol) release, after 1 and 3 mM sodium taurocholate stimulation, was higher in the mutant-transfected cell lines than that in the wild-type and was particularly enhanced in cells transfected with the p.Y403H-construct.In summary, our data show that both mutations do not seem to affect protein expression, but are able to reduce the efflux of phosphatidylcholine associated with increase of Chol, thereby promoting the formation of toxic bile.

Microgravity-driven remodeling of the proteome reveals insights into molecular mechanisms and signal networks involved in response to the space flight environment

UNLABELLED Space is a hostile environment characterized by high vacuum, extreme temperatures, meteoroids, space debris, ionospheric plasma, microgravity and space radiation, which all represent risks for human health. A deep understanding of the biological consequences of exposure to the space environment is required to design efficient countermeasures to minimize their negative impact on human health. Recently, proteomic approaches have received a significant amount of attention in the effort to further study microgravity-induced physiological changes. In this review, we summarize the current knowledge about the effects of microgravity on microorganisms (in particular Cupriavidus metallidurans CH34, Bacillus cereus and Rhodospirillum rubrum S1H), plants (whole plants, organs, and cell cultures), mammalian cells (endothelial cells, bone cells, chondrocytes, muscle cells, thyroid cancer cells, immune system cells) and animals (invertebrates, vertebrates and mammals). Herein, we describe their proteomes response to microgravity, focusing on proteomic discoveries and their future potential applications in space research. BIOLOGICAL SIGNIFICANCE Space experiments and operational flight experience have identified detrimental effects on human health and performance because of exposure to weightlessness, even when currently available countermeasures are implemented. Many experimental tools and methods have been developed to study microgravity induced physiological changes. Recently, genomic and proteomic approaches have received a significant amount of attention. This review summarizes the recent research studies of the proteome response to microgravity inmicroorganisms, plants, mammalians cells and animals. Current proteomic tools allow large-scale, high-throughput analyses for the detection, identification, and functional investigation of all proteomes. Understanding gene and/or protein expression is the key to unlocking the mechanisms behind microgravity-induced problems and to finding effective countermeasures to spaceflight-induced alterations but also for the study of diseases on earth. Future perspectives are also highlighted.