BackgroundThe 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/SignificanceDHA 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.

Neuropeptide Y (NPY) is a potent orexigenic neuropeptide implicated in feeding regulation in mammals. However, except for the case of the goldfish, the involvement of NPY in the feeding behaviour of teleost fish has not well been studied. Therefore, we investigated the role of NPY in food intake using a zebrafish (Danio rerio) model because the molecular bases of NPY and its receptor have been well studied in this species. We examined the effect of feeding status on NPY-like immunoreactivity and the expression level of the NPY transcript in the brain. The number of neuronal cells showing NPY-like immunoreactivity in the hypothalamic regions, including the periventricular nucleus of posterior tuberculum and the posterior tuberal nucleus, was significantly increased in fish fasted for 7 days. NPY mRNA levels in the hypothalamus, but not the telencephalon, obtained from fish fasted for 7 days were higher than those in fish that had been fed normally. We then investigated the effect of i.c.v. administration of NPY on food intake. Cumulative food intake was significantly increased by i.c.v. administration of NPY (at 1 and 10 pmol/g body weight; BW) during a 60-min observation period. The NPY-induced orexigenic action (at 10 pmol/g BW) was blocked by treatment with a NPY Y1 receptor antagonist, BIBP-3226, at 100 pmol/g BW. These results indicate that NPY acts as an orexigenic factor in the zebrafish.

Reactive oxygen metabolites, including free radicals such as nitric oxide (NO), superoxide anion (O 2 Ϫ ), hydroxyl ( · OH) radicals and peroxynitrite (ONOO Ϫ ), are toxic and play an important role in tissue injury.1,2) O 2 Ϫ reacts rapidly with NO to produce the more toxic ONOO Ϫ . In addition, ONOO Ϫ and its decomposition product, · OH, contribute to antioxidant depletion, alterations of protein structure and oxidative damage observed in human diseases. [3][4][5] Thus, it is important to find safe and effective scavengers of these oxygen radicals for prevention and treatment of oxidative stress-related diseases.Panax ginseng C. A. MEYER is one of the most widely used herbal medicines in the Orient. It has a wide range of pharmacological and physiological actions, such as antiaging, immunoenhancement, antistress and antitumor. [6][7][8] Of the two kinds of ginseng, white ginseng (WG), is air-dried ginseng, and red ginseng (RG) is produced by steaming raw ginseng at 98-100°C for 2-3 h. RG is reportedly more pharmacologically active than WG. These improved biological activities of ginseng result from changes in the chemical constituents that occur during steaming treatment. Ginseng saponins, referred to as ginsenosides, are believed to play a pharmacologically important role. Several investigators have reported new ginsenosides from RG that are not usually found in WG.9,10) Recently, a method which can enhance the yield of these RG specific ginsenosides by steaming ginseng at a temperature higher than RG has been developed.11) This heat-processed ginseng, termed sun ginseng (SG), has been reported to have more potent pharmacological activities, such as vasorelaxation, antioxidant and antitumor activities.12,13) In our preliminary study, SG also showed better NO-scavenging activity than conventional WG and RG. However, almost all ginsenosides had no effects on radical scavenging activity (data not shown). It is insufficient to explain the various pharmacological effects of ginseng with only ginsenosides, especially antioxidant activity related to radical scavenging. Therefore, in this study, we focused on phenolic compounds in ginseng, such as maltol, salicylic acid, vanillic acid and p-coumaric acid, known as principal antioxidant components of ginseng, to investigate the radical scavenging activity of SG. We examined the NO, O 2 Ϫ , ONOO Ϫ and · OHscavenging activities of WG, RG and SG. Furthermore, we also studied the radical scavenging activities of maltol, salicylic acid, vanillic acid and p-coumaric acid and the contents of these 4 phenolic compounds in WG, RG and SG. MATERIALS AND METHODSReagents Salicylic acid, p-coumaric acid, vanillic acid, maltol, sodium nitroprusside (SNP), nitro blue tetrazolium (NBT), b-nicotineamide adenine dinucleotide disodium salt reduced form (b-NADH), DL-penicillamine and L(ϩ)-ascorbic acid were obtained from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Bis(trimethylsilyl)trifluoroacetamide plus 1% trimethylchlorosilane, n-eicosane, phenazine methosulfate (PMS), 3-morpho...

In a recent study, we showed that eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), two common omega-3 fatty acids, can cause ROS accumulation and subsequently induce caspase-8-dependent apoptosis in human breast cancer cells (Kang et al. [2010], PLoS ONE 5: e10296). In this study, we showed that the pancreas has a unique ability to accumulate EPA at a level markedly higher than several other tissues analyzed. Based on this finding, we sought to further investigate the anticancer actions of EPA and its analog DHA in human pancreatic cancer cells using both in vitro and in vivo models. EPA and DHA were found to induce ROS accumulation and caspase-8-dependent cell death in human pancreatic cancer cells (MIA-PaCa-2 and Capan-2) in vitro. Feeding animals with a diet supplemented with 5% fish oil, which contains high levels of EPA and DHA, also strongly suppresses the growth of MIA-PaCa-2 human pancreatic cancer xenografts in athymic nude mice, by inducing oxidative stress and cell death. In addition, we showed that EPA can concomitantly induce autophagy in these cancer cells, and the induction of autophagy diminishes its ability to induce apoptotic cell death. It is therefore suggested that combination of EPA with an autophagy inhibitor may be a useful strategy in increasing the therapeutic effectiveness in pancreatic cancer.

AB. Neuronal gap junctions are required for NMDA receptormediated excitotoxicity: implications in ischemic stroke. J Neurophysiol 104: 3551-3556, 2010. First published October 13, 2010 doi:10.1152 doi:10. /jn.00656.2010 play an important role in cell survival versus cell death decisions during neuronal development, ischemia, trauma, and epilepsy. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all these conditions. In the developing CNS, neuronal gap junctions are critical for two different types of NMDAR-dependent cell death. However, whether neuronal gap junctions play a role in NMDAR-dependent neuronal death in the mature CNS was not known. Using Fluoro-Jade B staining, we show that a single intraperitoneal administration of NMDA (100 mg/kg) to adult wild-type mice induces neurodegeneration in three forebrain regions, including rostral dentate gyrus. However, the NMDARmediated neuronal death is prevented by pharmacological blockade of neuronal gap junctions (with mefloquine, 30 mg/kg) and does not occur in mice lacking neuronal gap junction protein, connexin 36. Using Western blots, electrophysiology, calcium imaging, and gas chromatography-mass spectrometry in wild-type and connexin 36 knockout mice, we show that the reduced level of neuronal death in knockout animals is not caused by the reduced expression of NMDARs, activity of NMDARs, or permeability of the blood-brain barrier to NMDA. In wild-type animals, this neuronal death is not caused by upregulation of connexin 36 by NMDA. Finally, pharmacological and genetic inactivation of neuronal gap junctions in mice also dramatically reduces neuronal death caused by photothrombotic focal cerebral ischemia. The results indicate that neuronal gap junctions are required for NMDAR-dependent excitotoxicity and play a critical role in ischemic neuronal death.

In recent years, several therapeutic drugs have been rationally designed and synthesized based on the novel knowledge gained from investigating the actions of biologically active chemicals derived from foods, plants, and medicinal herbs. One of the major advantages of these naturalistic chemicals is their ability to interact with multiple targets in the body resulting in a combined beneficial effect. Ginseng is a perennial herb (Araliaceae family), a species within the genus Panax, and a highly valued and popular medicinal plant. Evidence for the medicinal and health benefits of Panax ginseng and its components in preventing neurodegeneration has increased significantly in the past decade. The beneficial effects of P. ginseng on neurodegenerative diseases have been attributed primarily to the antioxidative and immunomodulatory activities of its ginsenoside components. Mechanistic studies on the neuroprotective effects of ginsenosides revealed that they act not only as antioxidants but also as modulators of intracellular neuronal signaling and metabolism, cell survival/death genes, and mitochondrial function. The goal of the present paper is to provide a brief review of recent knowledge and developments concerning the beneficial effects as well as the mechanism of action of P. ginseng and its components in the treatment and prevention of neurodegenerative diseases.

Diabetes is a disorder of excessive urine excretion and chronic hyperglycemia, and glucose participates in diabetic complications such as atherosclerosis, cardiac dysfunction, and nephropathy. Chronic hyperglycemia accelerates activation of the formation of advanced glycation end-products (AGEs), oxidative stress, the polyol pathway, and protein kinase C pathway. These metabolic factors are synergistically correlated with one another; therefore, an effective treatment with wide-spread effects continues to be required.Up to now, there have been many experiments focusing on the treatment of diabetes and its complications with traditional medicines including Chinese prescriptions because of their absence of toxic and/or side-effects. In our previous study, we reported that the Chinese prescriptions Hachimijio-gan and Keishi-bukuryo-gan had potential therapeutic effects against diabetic nephropathy, and had different functions in terms of their effects on metabolic disorders, especially on AGE formation in Hachimi-jio-gan and oxidative stress in Keishi-bukuryo-gan. [1][2][3][4] In addition, we also clarified that administration of dried Rehmanniae Radix (Rehmannia glutinosa LIBOSCH. var. purpurea MAKINO) extract, which is the main constituent of Hachimi-jio-gan, attenuates renal dysfunction in diabetic nephropathy mainly due to its suppression of oxidative stress 5) ; however, for the analysis of this prescription, further characterization of the other constituents is needed. According to the three-dimensional HPLC profile, as previously shown, 2,4) morroniside, loganin, and paeoniflorin were detected as the major compounds in Hachimi-jio-gan. Morroniside and loganin are the components of Corni Fructus (Cornus officinalis SIEB. et ZUCC.) and paeoniflorin is the component of Moutan Cortex (Paeonia suffruticosa ANDREWS) common in Keishi-bukuryogan. Therefore, in order to clarify the source of a particular action of Hachimi-jio-gan, we chose to evaluate the usefulness of one of the crude drugs, Corni Fructus.Corni Fructus has been used as a traditional medicine in Japan and China, and the components of this plant are iridoid total glycoside such as morroniside and loganin and also a few polyphenols such as cornusiin A, B, and C, monomeric and trimeric hydrolysable tannins, and so on.6,7) Recently, it has been reported that Corni Fructus has a plasma glucoselowering action in normal rats, along with anti-neoplastic and anti-microbial effects.8-10) Moreover, Vareed et al. 11) also reported that Corni Fructus has been used for improving liver and kidney functions, and iridoid total glycoside has the effect of preventing the overexpression of transforming growth factor (TGF)-b 1 and matrixes in glomeruli with a diabetic model. 12) However, the mechanisms of Corni Fructus against glucose-associated metabolic disorders in diabetes have yet to be explored. To determine whether Corni Fructus possesses the principal role in Hachimi-jio-gan, which has a strong effect on AGE formation in diabetes and/or diabetic nephropathy, we e...

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