A new colorimetric method for determining antioxidant levels using 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS).

We describe here a novel assay that determines the total a+ntioxidative activities of known antioxidants and antioxidants in beverages. The method employs the substrate 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) that yields the colored product 3,5,3',5'-tetrabromoazobenzene sulfate sodium salt (azo-TBBS). The amounts of azo-TBBS are measured using HPLC and then used to calculate total antioxidative capacity (TAC) values. We first show that the TAC values measured using the new DBNBS system were significantly higher compared with the control. The assay was validated through further analysis of 56 compounds, including previously characterized antioxidants. The data are consistent with published values. Here we describe in detail the application of the DBNBS method to the measurement of the TAC values of eight beverages, including wines and fruit juices. The DBNBS assay employs a readily applicable protocol that sensitively determines the levels of antioxidants in foodstuffs. - A new DBNBS-mediated antioxidant assay system is compared with standard DPPH and ORAC assays - DBNBS traps hydrogen radicals to generate a readily measured colored reduction product that quantifies antioxidant levels.

Selective Suppression of Cell Growth and Programmed Cell Death-Ligand 1 Expression in HT1080 Fibrosarcoma Cells by Low Molecular Weight Fucoidan Extract.

Low molecular weight fucoidan extract (LMF), prepared by an abalone glycosidase digestion of a crude fucoidan extracted from Cladosiphon novae-caledoniae Kylin, exhibits various biological activities, including anticancer effect. Various cancers express programmed cell death-ligand 1 (PD-L1), which is known to play a significant role in evasion of the host immune surveillance system. PD-L1 is also expressed in many types of normal cells for self-protection. Previous research has revealed that selective inhibition of PD-L1 expressed in cancer cells is critical for successful cancer eradication. In the present study, we analyzed whether LMF could regulate PD-L1 expression in HT1080 fibrosarcoma cells. Our results demonstrated that LMF suppressed PD-L1/PD-L2 expression and the growth of HT1080 cancer cells and had no effect on the growth of normal TIG-1 cells. Thus, LMF differentially regulates PD-L1 expression in normal and cancer cells and could serve as an alternative complementary agent for treatment of cancers with high PD-L1 expression.

An Exploratory Study on the Anti-inflammatory Effects of Fucoidan in Relation to Quality of Life in Advanced Cancer Patients.

BACKGROUND: Conventional anticancer therapies still cause difficulties with selective eradication and accompanying side effects that reduce patients' quality of life (QOL). Fucoidan is extracted from seaweeds and has already exhibited broad bioactivities, including anticancer and anti-inflammatory properties, in basic studies. It is expected to enhance therapeutic efficacy and minimize side effects in cancer patients; however, despite its potential benefits, there are very few clinical trials using fucoidans. Therefore, we performed an exploratory clinical study for advanced cancer patients to examine the efficacy of fucoidans, especially focusing on inflammation in relation to QOL scores. METHODS: We conducted a prospective, open-label clinical study for advanced cancer patients using fucoidans via oral administration; 20 advanced cancer patients with metastases were recruited and were given 400 mL/d fucoidan (10 mg/mL) for at least 4 weeks. Inflammatory biomarkers, including high-sensitivity C-reactive protein and various cytokines, and QOL scores were monitored before treatment, after 2 weeks, and after 4 weeks of fucoidan ingestion. RESULTS: The main proinflammatory cytokines, including interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α (TNF-α) were significantly reduced after 2 weeks of fucoidan ingestion. QOL scores, including fatigue, stayed almost stable without significant changes during the study period. The univariate and multivariate analyses revealed that the responsiveness of IL-1ß was a significant independent prognostic factor. CONCLUSION: This is the first study providing evidence of the anti-inflammatory effects of fucoidans for advanced cancer patients. In future studies, larger blinded, controlled trials are required to establish the efficacy of fucoidan as supportive care for cancer patients, especially those undergoing chemotherapy.

Electrochemically reduced water exerts superior reactive oxygen species scavenging activity in HT1080 cells than the equivalent level of hydrogen-dissolved water.

Electrochemically reduced water (ERW) is produced near a cathode during electrolysis and exhibits an alkaline pH, contains richly dissolved hydrogen, and contains a small amount of platinum nanoparticles. ERW has reactive oxygen species (ROS)-scavenging activity and recent studies demonstrated that hydrogen-dissolved water exhibits ROS-scavenging activity. Thus, the antioxidative capacity of ERW is postulated to be dependent on the presence of hydrogen levels; however, there is no report verifying the role of dissolved hydrogen in ERW. In this report, we clarify whether the responsive factor for antioxidative activity in ERW is dissolved hydrogen. The intracellular ROS scavenging activity of ERW and hydrogen-dissolved water was tested by both fluorescent stain method and immuno spin trapping assay. We confirm that ERW possessed electrolysis intensity-dependent intracellular ROS-scavenging activity, and ERW exerts significantly superior ROS-scavenging activity in HT1080 cells than the equivalent level of hydrogen-dissolved water. ERW retained its ROS-scavenging activity after removal of dissolved hydrogen, but lost its activity when autoclaved. An oxygen radical absorbance capacity assay, the 2,2-diphenyl-1-picrylhydrazyl assay and chemiluminescence assay could not detect radical-scavenging activity in both ERW and hydrogen-dissolved water. These results indicate that ERW contains electrolysis-dependent hydrogen and an additional antioxidative factor predicted to be platinum nanoparticles.

Electrochemically reduced water protects neural cells from oxidative damage.

Aging-related neurodegenerative disorders are closely associated with mitochondrial dysfunction and oxidative stresses and their incidence tends to increase with aging. Brain is the most vulnerable to reactive species generated by a higher rate of oxygen consumption and glucose utilization compared to other organs. Electrochemically reduced water (ERW) was demonstrated to scavenge reactive oxygen species (ROS) in several cell types. In the present study, the protective effect of ERW against hydrogen peroxide (H2O2) and nitric oxide (NO) was investigated in several rodent neuronal cell lines and primary cells. ERW was found to significantly suppress H2O2 (50-200 µM) induced PC12 and SFME cell deaths. ERW scavenged intracellular ROS and exhibited a protective effect against neuronal network damage caused by 200 µM H2O2 in N1E-115 cells. ERW significantly suppressed NO-induced cytotoxicity in PC12 cells despite the fact that it did not have the ability to scavenge intracellular NO. ERW significantly suppressed both glutamate induced Ca(2+) influx and the resulting cytotoxicity in primary cells. These results collectively demonstrated for the first time that ERW protects several types of neuronal cells by scavenging ROS because of the presence of hydrogen and platinum nanoparticles dissolved in ERW.

Removal efficiency of radioactive cesium and iodine ions by a flow-type apparatus designed for electrochemically reduced water production.

The Fukushima Daiichi Nuclear Power Plant accident on March 11, 2011 attracted people's attention, with anxiety over possible radiation hazards. Immediate and long-term concerns are around protection from external and internal exposure by the liberated radionuclides. In particular, residents living in the affected regions are most concerned about ingesting contaminated foodstuffs, including drinking water. Efficient removal of radionuclides from rainwater and drinking water has been reported using several pot-type filtration devices. A currently used flow-type test apparatus is expected to simultaneously provide radionuclide elimination prior to ingestion and protection from internal exposure by accidental ingestion of radionuclides through the use of a micro-carbon carboxymethyl cartridge unit and an electrochemically reduced water production unit, respectively. However, the removability of radionuclides from contaminated tap water has not been tested to date. Thus, the current research was undertaken to assess the capability of the apparatus to remove radionuclides from artificially contaminated tap water. The results presented here demonstrate that the apparatus can reduce radioactivity levels to below the detection limit in applied tap water containing either 300 Bq/kg of 137Cs or 150 Bq/kg of 125I. The apparatus had a removal efficiency of over 90% for all concentration ranges of radio-cesium and -iodine tested. The results showing efficient radionuclide removability, together with previous studies on molecular hydrogen and platinum nanoparticles as reactive oxygen species scavengers, strongly suggest that the test apparatus has the potential to offer maximum safety against radionuclide-contaminated foodstuffs, including drinking water.

Antigen-specific in vitro immunization: a source for human monoclonal antibodies.

Human monoclonal antibody has great potential for treatment of various diseases utilizing their specificity against antigens. We have shown an in vitro immunization (IVI) protocol inducing antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs) for efficient production of human monoclonal antibodies. By using IVI method antigen specific antibody genes can be efficiently obtained because of increasing production of antigen-specific antibodies from in vitro immunized PBMCs. This IVI protocol will be widely applied for combination with several display methods and enhance the production of human monoclonal antibodies.

Suppression of immunoglobulin production in human peripheral blood mononuclear cells by monocytes via secretion of heavy-chain ferritin.

In vitro antigen stimulation of peripheral blood mononuclear cells (PBMCs) does not induce immunoglobulin (Ig) production. However, pretreatment of PBMCs with l-leucyl-l-leucine methyl ester (LLME) prior to in vitro stimulation removes the suppression of Ig production. In the present study, we attempted to identify the target cells of LLME and determine the mechanisms by which Ig production in PBMCs is suppressed. We found that CD14(+) monocytes are involved in the suppression of Ig production in PBMCs. Furthermore, we confirmed that heavy-chain ferritin derived from CD14(+) monocytes suppresses Ig production in PBMCs, possibly through iron sequestration.

A rapid screening and production method using a novel mammalian cell display to isolate human monoclonal antibodies.

Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.

In vitro immunization of Epstein-Barr virus-immortalized B cells augments antigen-specific antibody production.

The current method for in vitro immunization (IVI) uses several antigens including toxins, food allergens, pathogenic bacteria, and self-antigen-derived peptides that induce an antigen-specific immune response in peripheral blood mononuclear cells (PBMCs). This protocol, however, requires donor blood collection and preparation of PBMCs before every IVI. In the present study, we aimed to design a more efficient system utilizing B cells immortalized with Epstein-Barr virus (EBV-B) as host cells for IVI to make antigen-specific antibodies. Results showed that previously antigen-sensitized, EBV-B cells exposed to the antigen along with IL-6, CpG oligonucleotides, and CD40 ligand signal produced antigen-specific antibodies. These results provide evidence for a novel and easy method to expand memory-type B cells and produce antigen-specific antibodies.

Induction of apoptosis by low-molecular-weight fucoidan through calcium- and caspase-dependent mitochondrial pathways in MDA-MB-231 breast cancer cells.

Fucoidan, a fucose-rich polysaccharide extracted from brown seaweed, has antitumor, anticoagulant, antiviral, anti-inflammatory, and antibacterial activities. Several studies have shown that a fucoidan treatment of cancer cells induced cytotoxicity and apoptosis and inhibited angiogenesis and invasion. We investigated in the present study the effect of low-molecular-weight fucoidan (LMWF) on apoptosis in estrogen receptor-negative MDA-MB-231 human breast cancer cells. The LMWF treatment of MDA-MB-231 cells was associated with the activation of caspases and mitochondrial dysfunction, including dissipation of the mitochondrial membrane potential (ΔΨm), alteration of Ca(2+) homeostasis, cytochrome c release, and decreased expression of antiapoptotic Bcl-2 family proteins. Understanding the molecular events that mediated LMWF-induced MDA-MB-231 cell death will contribute to a more rational approach to cancer chemotherapy.

Fucoidan extract enhances the anti-cancer activity of chemotherapeutic agents in MDA-MB-231 and MCF-7 breast cancer cells.

Fucoidan, a fucose-rich polysaccharide isolated from brown alga, is currently under investigation as a new anti-cancer compound. In the present study, fucoidan extract (FE) from Cladosiphon navae-caledoniae Kylin was prepared by enzymatic digestion. We investigated whether a combination of FE with cisplatin, tamoxifen or paclitaxel had the potential to improve the therapeutic efficacy of cancer treatment. These co-treatments significantly induced cell growth inhibition, apoptosis, as well as cell cycle modifications in MDA-MB-231 and MCF-7 cells. FE enhanced apoptosis in cancer cells that responded to treatment with three chemotherapeutic drugs with downregulation of the anti-apoptotic proteins Bcl-xL and Mcl-1. The combination treatments led to an obvious decrease in the phosphorylation of ERK and Akt in MDA-MB-231 cells, but increased the phosphorylation of ERK in MCF-7 cells. In addition, we observed that combination treatments enhanced intracellular ROS levels and reduced glutathione (GSH) levels in breast cancer cells, suggesting that induction of oxidative stress was an important event in the cell death induced by the combination treatments.

Protective effects of the fermented milk Kefir on X-ray irradiation-induced intestinal damage in B6C3F1 mice.

Gastrointestinal damage associated with radiation therapy is currently an inevitable outcome. The protective effect of Kefir was assessed for its usefulness against radiation-induced gastrointestinal damage. A Kefir supernatant was diluted by 2- or 10-fold and administered for 1 week prior to 8 Gray (Gy) X-ray irradiation at a dose rate of 2 Gy/min, with an additional 15 d of administration post-irradiation. The survival rate of control mice with normal drinking water dropped to 70% on days 4 through 9 post-irradiation. On the other hand, 100% of mice in the 10- and 2-fold-diluted Kefir groups survived up to day 9 post-irradiation (p<0.05 and p<0.01, respectively). Examinations for crypt regeneration against 8, 10 and 12 Gy irradiation at a dose rate of 4 Gy/min revealed that the crypt number was significantly increased in the mice administered both diluted Kefir solutions (p<0.01 for each). Histological and immunohistochemical examinations revealed that the diluted Kefir solutions protected the crypts from radiation, and promoted crypt regeneration. In addition, lyophilized Kefir powder was found to significantly recover the testis weights (p<0.05), but had no effects on the body and spleen weights, after 8 Gy irradiation. These findings suggest that Kefir could be a promising candidate as a radiation-protective agent.

Molecular hydrogen reduces LPS-induced neuroinflammation and promotes recovery from sickness behaviour in mice.

Molecular hydrogen has been shown to have neuroprotective effects in mouse models of acute neurodegeneration. The effect was suggested to be mediated by its free-radical scavenger properties. However, it has been shown recently that molecular hydrogen alters gene expression and protein phosphorylation. The aim of this study was to test whether chronic ad libitum consumption of molecular hydrogen-enriched electrochemically reduced water (H-ERW) improves the outcome of lipopolysaccharide (LPS)-induced neuroinflammation. Seven days after the initiation of H-ERW treatment, C57Bl/6 mice received a single injection of LPS (0.33 mg/kg i.p.) or an equivalent volume of vehicle. The LPS-induced sickness behaviour was assessed 2 h after the injection, and recovery was assessed by monitoring the spontaneous locomotor activity in the homecage for 72 h after the administration of LPS. The mice were killed in the acute or recovery phase, and the expression of pro- and antiinflammatory cytokines in the hippocampus was assessed by real-time PCR. We found that molecular hydrogen reduces the LPS-induced sickness behaviour and promotes recovery. These effects are associated with a shift towards anti-inflammatory gene expression profile at baseline (downregulation of TNF- α and upregulation of IL-10). In addition, molecular hydrogen increases the amplitude, but shortens the duration and promotes the extinction of neuroinflammation. Consistently, molecular hydrogen modulates the activation and gene expression in a similar fashion in immortalized murine microglia (BV-2 cell line), suggesting that the effects observed in vivo may involve the modulation of microglial activation. Taken together, our data point to the regulation of cytokine expression being an additional critical mechanism underlying the beneficial effects of molecular hydrogen.

Suppressive effects of electrochemically reduced water on matrix metalloproteinase-2 activities and in vitro invasion of human fibrosarcoma HT1080 cells.

It has been demonstrated that hydrogen peroxide (H(2)O(2)) is directly associated with elevated matrix metalloproteinase-2 (MMP-2) expression in several cell lines. Electrochemically reduced water (ERW), produced near the cathode during electrolysis, and scavenges intracellular H(2)O(2) in human fibrosarcoma HT1080 cells. RT-PCR and zymography analyses revealed that when HT1080 cells were treated with ERW, the gene expression of MMP-2 and membrane type 1 MMP and activation of MMP-2 was repressed, resulting in decreased invasion of the cells into matrigel. ERW also inhibited H(2)O(2)-induced MMP-2 upregulation. To investigate signal transduction involved in MMP-2 downregulation, mitogen-activated protein kinase (MAPK)-specific inhibitors, SB203580 (p38 MAPK inhibitor), PD98059 (MAPK/extracellular regulated kinase kinase 1 inhibitor) and c-Jun NH(2)-terminal kinase inhibitor II, were used to block the MAPK signal cascade. MMP-2 gene expression was only inhibited by SB203580 treatment, suggesting a pivotal role of p38 MAPK in regulation of MMP-2 gene expression. Western blot analysis showed that ERW downregulated the phosphorylation of p38 both in H(2)O(2)-treated and untreated HT1080 cells. These results indicate that the inhibitory effect of ERW on tumor invasion is due to, at least in part, its antioxidative effect.