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Autophagy is an evolutionarily conserved process to catabolize cytoplasmic proteins and organelles1, 2. During starvation, the target of rapamycin (TOR), a nutrient-responsive kinase, is inhibited, thereby inducing autophagy. In autophagy, double-membrane autophagosomes envelop and sequester intracellular components and then fuse with lysosomes to form autolysosomes which degrade their contents to regenerate nutrients. Current models of autophagy terminate with the degradation of autophagosome cargo in autolysosomes3-5, but the regulation of autophagy in response to nutrients and the subsequent fate of the autolysosome are poorly defined. Here we show that mTOR signaling is inhibited during autophagy initiation, but reactivated with prolonged starvation. mTOR reactivation is autophagy-dependent, and requires the degradation of autolysosomal products. Increased mTOR activity attenuates autophagy and generates proto-lysosomal tubules and vesicles that extrude from autolysosomes and ultimately mature into functional lysosomes, thereby restoring the full complement of lysosomes in the cell – a process we identify in multiple animal species. Thus, an evolutionarily-conserved cycle in autophagy governs nutrient sensing and lysosome homeostasis during starvation.

The docosahexaenoic (DHA), a ω-3 fatty acid, could play a beneficial inhibition of the incidence and progress of a series of human diseases including cancer. It has been report that DHA is involved in cell apoptosis. Recent studies show that the signal transduction pathway links with bcl-2, bax, caspase-3 and MMP-9 molecules. Therefore, we tested the relationship between DHA and cell apoptosis in human hepatocellular carcinoma cells (Bel-7402 cells). We show here that DHA induces Bel-7402 cells apoptosis after pre-treating cells with DHA. DHA down-regulates the protein expression of Bcl-2 and Bim mRNA level, and up-regulates caspase-3 activity and Bax expression level. We also found that DHA inhibits Bel-7402 cells migration. Basic on our studies, DHA may play a role in tumor invasion and survival.

Docosahexaenoic acid (DHA), an omega-3 polyunsaturated fatty acid, is an essential component of membrane phosphatides and has been implicated in cognitive functions. Low levels of circulating or brain DHA are associated with various neurocognitive disorders including Alzheimer’s disease (AD), while laboratory animals, including animal models of AD, can exhibit improved cognitive ability with a diet enriched in DHA. Various cellular mechanisms have been proposed for DHA’s behavioral effects, including increases in cellular membrane fluidity, promotion of neurite extension, and inhibition of apoptosis. However, there is little direct evidence that DHA affects synaptic structure in living animals. Here we show that oral supplementation with DHA substantially increases the number of dendritic spines in adult gerbil hippocampus, particularly when animals are co-supplemented with a uridine source, uridine-5’-monophosphate (UMP), which increases brain levels of the rate-limiting phosphatide precursor CTP. The increase in dendritic spines (> 30%) is accompanied by parallel increases in membrane phosphatides, and in pre- and post-synaptic proteins within the hippocampus. Hence oral DHA may promote neuronal membrane synthesis to increase the number of synapses, particularly when co-administered with UMP. Our findings provide a possible explanation for the effects of DHA on behavior and also suggest a strategy to treat cognitive disorders resulting from synapse loss.

The role of autophagy in oncogenesis remains ambiguous, and mechanisms that induce autophagy and regulate its outcome in human cancers are poorly understood. The maternally imprinted Ras-related tumor suppressor gene aplasia Ras homolog member I (ARHI; also known as DIRAS3) is downregulated in more than 60% of ovarian cancers, and here we show that re-expression of ARHI in multiple human ovarian cancer cell lines induces autophagy by blocking PI3K signaling and inhibiting mammalian target of rapamycin (mTOR), upregulating ATG4, and colocalizing with cleaved microtubule-associated protein light chain 3 (LC3) in autophagosomes. Furthermore, ARHI is required for spontaneous and rapamycin-induced autophagy in normal and malignant cells. Although ARHI re-expression led to autophagic cell death when SKOv3 ovarian cancer cells were grown in culture, it enabled the cells to remain dormant when they were grown in mice as xenografts. When ARHI levels were reduced in dormant cells, xenografts grew rapidly. However, inhibition of ARHI-induced autophagy with chloroquine dramatically reduced regrowth of xenografted tumors upon reduction of ARHI levels, suggesting that autophagy contributed to the survival of dormant cells. Further analysis revealed that autophagic cell death was reduced when cultured human ovarian cancer cells in which ARHI had been re-expressed were treated with growth factors (IGF-1, M-CSF), angiogenic factors (VEGF, IL-8), and matrix proteins found in xenografts. Thus, ARHI can induce autophagic cell death, but can also promote tumor dormancy in the presence of factors that promote survival in the cancer microenvironment.

The epidermal growth factor receptor (EGFR), which regulates cell growth and survival, is integral to colon tumorigenesis. Lipid rafts play a role in regulating EGFR signaling, and docosahexaenoic acid (DHA) is known to perturb membrane domain organization through changes in lipid rafts. Therefore, we investigated the mechanistic link between EGFR function and DHA. Membrane incorporation of DHA into immortalized colonocytes altered the lateral organization of EGFR. DHA additionally increased EGFR phosphorylation but paradoxically suppressed downstream signaling. Assessment of the EGFR-Ras-ERK1/2 signaling cascade identified Ras GTP binding as the locus of the DHA-induced disruption of signal transduction. DHA also antagonized EGFR signaling capacity by increasing receptor internalization and degradation. DHA suppressed cell proliferation in an EGFR-dependent manner, but cell proliferation could be partially rescued by expression of constitutively active Ras. Feeding chronically-inflamed, carcinogen-injected C57BL/6 mice a fish oil containing diet enriched in DHA recapitulated the effects on the EGFR signaling axis observed in cell culture and additionally suppressed tumor formation. We conclude that DHA-induced alteration in both the lateral and subcellular localization of EGFR culminates in the suppression of EGFR downstream signal transduction, which has implications for the molecular basis of colon cancer prevention by DHA.

Autophagy is a regulated catabolic process triggered in cells deprived of nutrients or growth factors that govern nutrient uptake. Here we report that autophagy is induced by cetuximab, a therapeutic antibody that blocks EGFR function. Cancer cell treatment with cetuximab triggered autophagosome formation, conversion of microtubule-associated protein 1 light chain 3 from its cytoplasmic to membrane-associated form, and increased acidic vesicular organelle formation. Autophagy occurred when cetuximab inhibited the class I PI3K/Akt/mTOR pathway, but not when it inhibited only the MEK/Erk pathway, and it was accompanied by decreased levels of hypoxia inducible factor-1 alpha (HIF-1α) and Bcl-2. Stable overexpression of a HIF-1α mutant prevented cetuximab-induced autophagy and decrease in Bcl-2 levels. Knockdown of autophagy regulator beclin 1 or cell treatment with autophagy inhibitor 3-methyladenine, a class III PI3K (hVps34) inhibitor, also inhibited cetuximab-induced autophagy. Furthermore, knockdown of beclin 1 or Atg7 or treatment with the lysosome inhibitor chloroquine sensitized cancer cells to cetuximab-induced apoptosis. Mechanistic analysis argued that cetuximab acted by promoting an association between beclin 1 and hVps34, which was inhibited by overexpression of Bcl-2. Our findings suggest that the autophagy protects cancer cells from the pro-apoptotic effects of cetuximab.

Calcium can play an important role in the regulation of autophagy. We previously reported that exogenously introduced calcium in the form of calcium phosphate precipitates (CPP) induces autophagy. Here we showed that CPP-induced autophagy required the classical autophagic machinery, including the autophagosome initiating molecules FIP200 and Beclin 1, as well as molecules involved in the autophagosome membrane extension, Atg4, Atg5 and Atg3. On the other hand, Atg9 seemed to place a restriction on CPP-induced autophagy. Loss of Atg9 led to enhanced LC3 punctation and enhanced p62 degradation. CPP-induced autophagy was independent of mTOR and reactive oxygen species. It also did not affect MAP kinase activation and ER stress. DFCP1 is an ER-resident molecule that binds to phosphatidylinositol 3-phosphate. CPP activated DFCP1 punctation in a class III phosphatidylinositol-3-kinase and calcium dependent manner, and caused the association of DFCP1 puncta with the autophagosomes. Consistently, ER membranes, but not Golgi or mitochondrial membranes, colocalized with CPP-induced LC3 positive autophagosomes. These data suggest that CPP-induced autophagosome formation involves the interaction with the ER membrane.

Mitochondria can depolarize and trigger cell death through the opening of the mitochondrial permeability transition pore (MPTP). We recently showed that an increase in the long chain n3 polyunsaturated fatty acids (PUFA) docosahexaenoic acid (DHA; 22:6n3) and depletion of the n6 PUFA arachidonic acid (ARA; 20:4n6) in mitochondrial membranes is associated with a greater Ca2+ load required to induce MPTP opening. Here we manipulated mitochondrial phospholipid composition by supplementing the diet with DHA, ARA or combined DHA+ARA in rats for 10 weeks. There were no effects on cardiac function, or respiration of isolated mitochondria. Analysis of mitochondrial phospholipids showed DHA supplementation increased DHA and displaced ARA in mitochondrial membranes, while supplementation with ARA or DHA+ARA increased ARA and depleted linoleic acid (18:2n6). Phospholipid analysis revealed a similar pattern, particularly in cardiolipin. Tetralinoleoyl cardiolipin was depleted by 80% with ARA or DHA+ARA supplementation, with linoleic acid side chains replaced by ARA. Both the DHA and ARA groups had delayed Ca2+-induced MPTP opening, but the DHA+ARA group was similar to the control diet. In conclusion, alterations in mitochondria membrane phospholipid fatty acid composition caused by dietary DHA or ARA was associated with a greater cumulative Ca2+ load required to induced MPTP opening. Further, high levels of tetralinoleoyl cardiolipin were not essential for normal mitochondrial function if replaced with very-long chain n3 or n6 PUFAs.

Autophagy is a highly regulated intracellular process involved in the turnover of most cellular constituents and in the maintenance of cellular homeostasis. In this study, we show that the activity of autophagy increases in H2O2 or RasV12-induced senescent fibroblasts. Inhibiting autophagy promotes cell apoptosis in senescent cells, suggesting that autophagy activation plays a cytoprotective role. Furthermore, our data indicate that the increase of autophagy in senescent cells is linked to the activation of transcription factor FoxO3A, which blocks ATP generation by transcriptionally up-regulating the expression of PDK4, an inhibitor of pyruvate dehydrogenase complex, thus leading to AMPK activation and mTOR inhibition. These findings suggest a novel mechanism by which FoxO3A factors can activate autophagy via metabolic alteration.

The apoptotic effects of docosahexaenoic acid (DHA) and other ω-3 polyunsaturated fatty acids (PUFAs) have been documented in cell and animal studies. The molecular mechanism by which DHA induces apoptosis is unclear. Although there is no direct evidence, some studies have suggested that DNA damage generated through lipid peroxidation may be involved. Our previous studies showed that DHA, because it is high degree of unsaturation, can give rise to the acrolein-derived 1,N2-propanodeoxyguanosine (Acr-dG) as a major class of DNA adducts via lipid oxidation. As a first step to investigate the possible role of oxidative DNA damage in apoptosis induced by DHA, we examined the relationships between oxidative DNA damage and apoptosis caused by DHA in human colon cancer HT-29 cells. The apoptosis and oxidative DNA damage, including Acr-dG and 8-oxo-deoxyguanosine (8-oxo-dG) formation, in cells treated with DHA and ω-6 PUFAs, including arachidonic acid (AA) and linoleic acid (LA), were measured. DHA induced apoptosis in a dose- and time-dependent manner with a concentration range from 0 to 300 µM as indicated by increased caspase-3 activity and PARP cleavage. In contrast, AA and LA had little or no effect at these concentrations. The Acr-dG levels were increased in HT-29 cells treated with DHA at 240 and 300µM, and the increases were correlated with the induction of apoptosis at these concentrations, while no significant changes were observed for 8-oxo-dG. Because proteins may compete with DNA to react with Acr, we then examined the effects of BSA on the DHA induced apoptosis and oxidative DNA damage. The addition of BSA to HT-29 cell culture media significantly decreases Acr-dG levels with a concomitant decrease in the apoptosis induced by DHA. The reduced Acr-dG formation is attributed to the reaction of BSA with acrolein as indicated by increased levels of total protein carbonyls. Similar correlations between Acr-dG formation and apoptosis were observed in HT-29 cells directly incubated with 0 to 200µM of acrolein. Additionally, DHA treatment increased level of DNA strand breaks and caused cell cycle arrested at G1 phase. Taken together, these results demonstrate the parallel relationships between the Acr-dG level and apoptosis in HT-29 cells, suggesting that the formation of Acr-dG in cellular DNA may contribute to apoptosis induced by DHA.

Liver cancer ranks in prevalence and mortality among top five cancers worldwide. Accumulating interests have been focused in developing new strategies for liver cancer treatment. We have previously showed that dihydroartemisinin (DHA) exhibited antitumor activity towards liver cancer. In this study, we demonstrated that histone deacetylase inhibitors (HDACi) significantly augmented the antineoplastic effect of DHA via increasing apoptosis in vitro and in vivo. Inhibition of ERK phosphorylation contributed to DHA-induced apoptosis, due to the fact that inhibitor of ERK phosphorylation (PD98059) increased DHA-induced apoptosis. Compared with DHA alone, the combined treatment with DHA and HDACi reduced mitochondria membrane potential, released cytochrome c into cytoplasm, increased p53 and Bak, decreased Mcl-1 and p-ERK, activated caspase 3 and PARP, and induced apoptotic cells. Furthermore, we showed that HDACi pretreatment facilitated DHA-induced apoptosis. In Hep G2-xenograft carrying nude mice, the intraperitoneal injection of DHA and SAHA resulted in significant inhibition of xenograft tumors. Results of TUNEL and H&E staining showed more apoptosis induced by combined treatment. Immunohistochemistry data revealed the activation of PARP, and the decrease of Ki-67, p-ERK and Mcl-1. Taken together, our data suggest that the combination of HDACi and DHA offers an antitumor effect on liver cancer, and this combination treatment should be considered as a promising strategy for chemotherapy.

Dietary intake of long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) has been reported to decrease several markers of lymphocyte activation and modulate monocyte susceptibility to apoptosis. However most human studies examined the combined effect of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) using relatively high daily amounts of n-3 PUFA. The present study investigated the effects of increasing doses of DHA added to the regular diet of human healthy volunteers on lymphocyte response to tetradecanoylphorbol acetate (TPA) plus ionomycin activation, and on monocyte apoptosis induced by oxidized LDL (oxLDL). Eight subjects were supplemented with increasing daily doses of DHA (200, 400, 800 and 1600mg) in a triacylglycerol form containing DHA as the only PUFA, for two weeks each dose. DHA intake dose-dependently increased the proportion of DHA in mononuclear cell phospholipids, the augmentation being significant after 400mg DHA/day. The TPA plus ionomycin-stimulated IL-2 mRNA level started to increase after ingestion of 400mg DHA/day, with a maximum after 800mg intake, and was positively correlated (P<0.003) with DHA enrichment in cell phospholipids. The treatment of monocytes by oxLDL before DHA supplementation drastically reduced mitochondrial membrane potential as compared with native LDL treatment. OxLDL apoptotic effect was significantly attenuated after 400mg DHA/day and the protective effect was maintained throughout the experiment, although to a lesser extent at higher doses. The present results show that supplementation of the human diet with low DHA dosages improves lymphocyte activability. It also increases monocyte resistance to oxLDL-induced apoptosis, which may be beneficial in the prevention of atherosclerosis.

The goal of the study is to examine the relationship between the sensor molecules, Hypoxia Inducible Factor-1 (HIF-1), AMP activated Protein Kinase (AMPK) and mammalian Target of Rapamycin (mTOR) in chondrocyte survival and autophagy. We showed that chondrocytes expressed the energy sensor AMPK-1 and that activation increased with maturation. In addition, we showed that thapsigargin treatment activated AMPK and autophagy in a HIF-1 dependent manner. Using serum-starved AMPK-silenced cells, we demonstrated that AMPK was required for the induction of the autophagic response. We also noted a change in chondrocyte sensitivity to apoptogens, due to activation of caspase-8 and cleavage and activation of the pro-apoptotic protein, BID. To test the hypothesis that AMPK signaling directly promoted autophagy, we inhibited AMPK activity in mTOR silenced cells and showed that while mTOR suppression induced autophagy, AMPK inhibition did not block this activity. Based on these findings, it is concluded that due to the micro-environmental changes experienced by the chondrocyte, autophagy is activated by AMPK in a HIF-1 dependent manner.

During the lipid peroxidation reaction, lipid hydroperoxides are formed as primary products. Several lines of evidence suggest that lipid hydroperoxides can trigger cell death in many cell types, including neurons. In a screening of lipid hydroperoxides which can induce toxicity in neuronal cells, we found docosahexaenoic acid hydroperoxides (DHA-OOH) induced much severe levels of reactive oxygen species generation and cell death in human neuroblastoma SH-SY5Y cells compared to the hydroperoxides of linoleic acid and arachidonic acid. Therefore, we focused on DHA-OOH, and demonstrated that DHA-OOH apparently induced an apoptosis in the neuronal cells through several apoptotic hallmarks including nuclei condensation, DNA fragmentation, poly (ADP-ribose) polymerase cleavage and increased activity of caspase-3. We also found the signaling changes in mitochondria-mediated apoptosis, such as cytochrome c release and increased expression of Bcl-2, as well as a dose-dependent attenuation of mitochondrial membrane potential in the DHA-OOH treated cells. These data indicated DHA hydroperoxide as a potential inducer of apoptosis in human neuroblastoma SH-SY5Y cells, which may be mediated by mitochondria dysfunction pathway.

Autophagy delivers cytoplasmic constituents to autophagosomes and is involved in innate and adaptive immunity. Cytosolic phospholipase (cPLA2) initiated pro-inflammatory lipid mediator pathways play a critical role in host defense and inflammation. The crosstalk between the two pathways remains unclear. Here, we report that cPLA2 and its metabolite lipid mediators induced autophagy in the RAW246.7 macrophage cell line and in primary monocytes. IFN-γ triggered autophagy involves activation of cPLA2. Cysteinyl leukotrienes (CysLTs) D4 and E4 and Prostagladin D2 (PGD2) also induced these effects. The autophagy is independent of changes in mTOR or autophagic flux. cPLA2 and lipid mediator-induced autophagy is ATG5 dependent. These data suggest that lipid mediators play a role in the regulation of autophagy, demonstrating a connection between the two seemingly separate innate immune responses, induction of autophagy and lipid mediator generation.

Autophagy is a cellular process that sequesters cargo in double-membraned vesicles termed autophagosomes and delivers this cargo to lysosomes to be degraded. It is enhanced during nutrient starvation to increase the rate of amino acid turnover. Diverse roles for autophagy have been reported for viral infections, including the assembly of viral replication complexes on autophagic membranes and protection of host cells from cell death. Here, we show that autophagosomes accumulate in Semliki Forest virus (SFV)-infected cells. Despite this, disruption of autophagy had no effect on the viral replication rate or formation of viral replication complexes. Also, viral proteins rarely colocalized with autophagosome markers, suggesting that SFV did not utilize autophagic membranes for its replication. Further, we found that SFV infection, unlike nutrient starvation, did not inactivate the constitutive negative regulator of autophagosome formation, mammalian target of rapamycin, suggesting that SFV-dependent accumulation of autophagosomes was not a result of enhanced autophagosome formation. In starved cells, addition of NH4Cl, an inhibitor of lysosomal acidification, caused a dramatic accumulation of starvation-induced autophagosomes, while in SFV-infected cells, NH4Cl did not further increase levels of autophagosomes. These results suggest that accumulation of autophagosomes in SFV-infected cells is due to an inhibition of autophagosome degradation rather than enhanced rates of autophagosome formation. Finally, we show that the accumulation of autophagosomes in SFV-infected cells is dependent on the expression of the viral glycoprotein spike complex.

Apoptotic cell death is mediated by caspase activation. Autophagy involves the sequestration of cytoplasmic contents into autophagosomes for traffic to lysosomes for degradation. While autophagy is antiapoptotic, increased numbers of autophagosomes have been associated with forms of non-apoptotic cell death. Apoptosis and autophagy may be co-regulated in the same directions, as the anti-apoptotic Bcl-2 and Bcl-xL proteins negatively regulate autophagy by binding to Beclin 1 (mammalian Atg6), and pro-apoptotic BH3-only proteins may reverse this effect by displacing these interactions. Here we show that apoptosis can suppress autophagy. Apoptosis induced by the pro-apoptotic protein Bax reduced autophagy by enhancing caspase-mediated cleavage of Beclin 1 at D149. After cleavage, both N and C-terminal Beclin 1 fragments change their localisations and these fragments do not interact normally with Vps34, which is required for autophagy. The cleavage of Beclin 1 is a critical event whereby caspases inhibit autophagy, as a non-cleavable Beclin 1 mutant restored autophagy in cells overexpressing Bax.

Autophagy is an intracellular degradation system, by which cytoplasmic contents are degraded in lysosomes. Autophagy is dynamically induced by nutrient depletion to provide necessary amino acids within cells, thus helping them adapt to starvation. Although it has been suggested that mTOR is a major negative regulator of autophagy, how it controls autophagy has not yet been determined. Here, we report a novel mammalian autophagy factor, Atg13, which forms a stable ∼3-MDa protein complex with ULK1 and FIP200. Atg13 localizes on the autophagic isolation membrane and is essential for autophagosome formation. In contrast to yeast counterparts, formation of the ULK1–Atg13–FIP200 complex is not altered by nutrient conditions. Importantly, mTORC1 is incorporated into the ULK1–Atg13–FIP200 complex through ULK1 in a nutrient-dependent manner and mTOR phosphorylates ULK1 and Atg13. ULK1 is dephosphorylated by rapamycin treatment or starvation. These data suggest that mTORC1 suppresses autophagy through direct regulation of the ∼3-MDa ULK1–Atg13–FIP200 complex.

Numerous reports have documented the beneficial effects of dietary docosahexaenoic acid (DHA) on beta-amyloid production and Alzheimer's disease (AD). However, none of these studies have examined and compared DHA, in combination with other dietary nutrients, for its effects on plaque pathogenesis. Potential interactions of DHA with other dietary nutrients and fatty acids are conventionally ignored. Here we investigated DHA with two dietary regimes; peptamen (pep+DHA) and low fat diet (low fat+DHA). Peptamen base liquid diet is a standard sole-source nutrition for patients with gastrointestinal dysfunction. Here we demonstrate that a robust AD transgenic mouse model shows an increased tendency to produce beta-amyloid peptides and amyloid plaques when fed a pep+DHA diet. The increase in beta-amyloid peptides was due to an elevated trend in the levels of beta-secretase amyloid precursor protein (APP) cleaving enzyme (BACE), the proteolytic C-terminal fragment beta of APP and reduced levels of insulin degrading enzyme that endoproteolyse beta-amyloid. On the contrary, TgCRND8 mice on low fat+DHA diet (based on an approximately 18% reduction of fat intake) ameliorate the production of abeta peptides and consequently amyloid plaques. Our work not only demonstrates that DHA when taken with peptamen may have a tendency to confer a detrimental affect on the amyloid plaque build up but also reinforces the importance of studying composite lipids or nutrients rather than single lipids or nutrients for their effects on pathways important to plaque development.

Cadmium is a toxic heavy metal which is environmentally and occupationally relevant. The mechanisms underlying cadmium-induced autophagy are not yet completely understood. The present study shows that cadmium induces autophagy, as demonstrated by the increase of LC3-II formation and the GFP-LC3 puncta cells. The induction of autophagosomes was directly visualized by electron microscopy in cadmium-exposed skin epidermal cells. Blockage of LKB1 or AMPK by siRNA transfection suppressed cadmium-induced autophagy. Cadmium-induced autophagy was inhibited in dominant-negative AMPK-transfected cells, whereas it was accelerated in cells transfected with the constitutively active form of AMPK. mTOR signaling, a negative regulator of autophagy, was downregulated in cadmium-exposed cells. In addition, cadmium generated reactive oxygen species (ROS) at relatively low levels, and caused poly(ADP-ribose) polymerase-1 (PARP) activation and ATP depletion. Inhibition of PARP by pharmacological inhibitors or its siRNA transfection suppressed ATP reduction and autophagy in cadmium-exposed cells. Furthermore, cadmium-induced autophagy signaling was attenuated by either exogenous addition of catalase and superoxide dismutase, or by overexpression of these enzymes. Consequently, these results suggest that cadmium-mediated ROS generation causes PARP activation and energy depletion, and eventually induces autophagy through the activation of LKB1-AMPK signaling and the down-regulation of mTOR in skin epidermal cells.

Exposure to ultraviolet-B (UVB) radiation induces inflammation and photocarcinogenesis in mammalian skin. Docosahexaenoic acid (DHA), a representative ω-3 polyunsaturated fatty acid, has been reported to possess anti-inflammatory and chemopreventive properties. In the present study, we investigated the molecular mechanisms underlying the inhibitory effects of DHA on UVB-induced inflammation in mouse skin. Our study revealed that topical application of DHA prior to UVB irradiation attenuated the expression of cyclooxygenase-2 (COX-2) and NAD(P)H:oxidase-4 (NOX-4) in hairless mouse skin. DHA pretreatment also attenuated UVB-induced DNA binding of nuclear factor-kappaB (NF-κB) through the inhibition of phosphorylation of IκB kinase-α/β, phosphorylation and degradation of IκBα and nuclear translocation of p50 and p65. In addition, UVB-induced phosphorylation of p65 at the serine 276 residue was significantly inhibited by topical application of DHA. Irradiation with UVB induced phosphorylation of mitogen and stress-activated kinase-1 (MSK1), extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase, and all these events were attenuated by pretreatment with DHA. Blocking ERK and p38 MAP kinase signaling by U0126 and SB203580, respectively, diminished MSK1 phosphorylation in UVB-irradiated mouse skin. Pretreatment with H-89, a pharmacological inhibitor of MSK1, abrogated UVB-induced activation of NF-κB and the expression of COX-2 and NOX-4 in mouse skin. In conclusion, topically applied DHA inhibits the UVB-induced activation of NF-κB and the expression of COX-2 and NOX-4 by blocking the phosphorylation of MSK1, a kinase downstream of ERK and p38 MAP kinase, in hairless mouse skin.

Autophagy is a transport system mediated by vesicles, ubiquitous in eukaryotic cells, by which bulk cytoplasm is targeted to a lysosome or vacuole for degradation. In the yeast Saccharomyces cerevisiae, autophagy is triggered by nutritional stress conditions (e.g., carbon- or nitrogen-depleted medium). In this study we showed that there is induction of autophagy in second-fermentation yeasts during sparkling wine making. Two methods were employed to detect autophagy: a biochemical approach based on depletion of the protein acetaldehyde dehydrogenase Ald6p and a morphological strategy consisting of visualization of autophagic bodies and autophagosomes, which are intermediate vesicles in the autophagic process, by transmission electron microscopy. This study provides the first demonstration of autophagy in second-fermentation yeasts under enological conditions. The correlation between autophagy and yeast autolysis during sparkling wine production is discussed, and genetic engineering of autophagy-related genes in order to accelerate the aging steps in wine making is proposed.

Background

The present study sought to further investigate the in vitro and in vivo anticancer effects of a representative omega-3 fatty acid, docosahexaenoic acid (DHA), with a focus on assessing the induction of oxidative stress and apoptosis as an important mechanism for its anticancer actions.

Methodology/Principal Findings

In vitro studies showed that DHA strongly reduces the viability and DNA synthesis of MCF-7 human breast cancer cells in culture, and also promotes cell death via apoptosis. Mechanistically, accumulation of reactive oxygen species and activation of caspase 8 contribute critically to the induction of apoptotic cell death. Co-presence of antioxidants or selective inhibition or knockdown of caspase 8 each effectively abrogates the cytotoxic effect of DHA. Using athymic nude mice as an in vivo model, we found that feeding animals the 5% fish oil-supplemented diet for 6 weeks significantly reduces the growth of MCF-7 human breast cancer cells in vivo through inhibition of cancer cell proliferation as well as promotion of cell death. Using 3-nitrotyrosine as a parameter, we confirmed that the fish oil-supplemented diet significantly increases oxidative stress in tumor cells in vivo. Analysis of fatty acid content in plasma and tissues showed that feeding animals a 5% fish oil diet increases the levels of DHA and eicosapentaenoic acid in both normal and tumorous mammary tissues by 329% and 300%, respectively.

Conclusions/Significance

DHA can strongly induce apoptosis in human MCF-7 breast cancer cells both in vitro and in vivo. The induction of apoptosis in these cells is selectively mediated via caspase 8 activation. These observations call for further studies to assess the effectiveness of fish oil as a dietary supplement in the prevention and treatment of human breast cancer.

Accumulating evidence suggests that the pathophysiology of depression might be associated with neuroinflammation, which could be attenuated by pharmacological treatment for depression. Omega-3 polyunsaturated fatty acids (PUFAs) are anti-inflammatory and exert antidepressant effects. The aim of this study was to identify the molecular mechanisms through which docosahexaenoic acid (DHA), the main omega-3 PUFA in the brain, modulates oxidative reactions and inflammatory cytokine production in microglial and neuronal cells. The results of this study showed that DHA reduced expressions of tumor necrosis factor-α, interleukin-6, nitric oxide synthase, and cyclo-oxygenase-2, induced by interferon-γ, and induced upregulation of heme oxygenase-1 (HO-1) in BV-2 microglia. The inhibitory effect of DHA on nitric oxide production was abolished by HO-1 inhibitor zinc protoporphyrin IX. In addition, DHA caused AKT and ERK activation in a time-dependent manner, and the DHA-induced HO-1 upregulation could be attenuated by PI-3 kinase/AKT and MEK/ERK inhibitors. DHA also increased IKKα/β phosphorylation, IκBα phosphorylation, and IκBα degradation, whereas both nuclear factor-κB and IκB protease inhibitors could inhibit DHA-induced HO-1 expressions. The other major n-3 PUFA, eicosapentaenoic acid, showed similar effects of DHA on inflammation and HO-1 in repeated key experiments. In connecting with inflammation hypothesis of depression and clinical studies supporting the antidepressant effects of omega-3 PUFAs, this study provides a novel implication of the antidepressant mechanisms of DHA.

Background/Aims

We reported that reduced dietary intake of polyunsaturated fatty acids (PUFA) such as arachidonic (AA,20:4n6, omega-6) and docosahexaenoic (DHA,22:6n3, omega-3) acids led to alcohol-induced fatty liver and fibrosis. This study was aimed at studying the mechanisms by which a DHA/AA-supplemented diet prevents alcohol-induced fatty liver.

Methods

Male Long-Evans rats were fed an ethanol or control liquid-diet with or without DHA/AA for 9 weeks. Plasma transaminase levels, liver histology, oxidative/nitrosative stress markers, and activities of oxidatively-modified mitochondrial proteins were evaluated.

Results

Chronic alcohol administration increased the degree of fatty liver but fatty liver decreased significantly in rats fed the alcohol-DHA/AA-supplemented diet. Alcohol exposure increased oxidative/nitrosative stress with elevated levels of ethanol-inducible CYP2E1, nitric oxide synthase, nitrite and mitochondrial hydrogen peroxide. However, these increments were normalized in rats fed the alcohol-DHA/AA-supplemented diet. The number of oxidatively-modified mitochondrial proteins was markedly increased following alcohol exposure but significantly reduced in rats fed the alcohol-DHA/AA-supplemented diet. The suppressed activities of mitochondrial aldehyde dehydrogenase, ATP synthase, and 3-ketoacyl-CoA thiolase in ethanol-exposed rats were also recovered in animals fed the ethanol-DHA/AA-supplemented diet.

Conclusions

Addition of DHA/AA prevents alcohol-induced fatty liver and mitochondrial dysfunction in an animal model by protecting various mitochondrial enzymes most likely through reducing oxidative/nitrosative stress.