Red Ginseng Extract and γ-Aminobutyric Acid Synergistically Enhance Immunity Against Cancer Cells and Antitumor Metastasis Activity in Mice.

The effects of combined administration of red ginseng (RG) extracts and gamma-aminobutyric acid (GABA) on immunostimulatory activity and tumor metastasis inhibition were investigated in mice. For the immunostimulatory activity, splenocyte proliferation, natural killer (NK) cell activity, including the production of granzyme B (GrB) and interferon gamma (IFN-γ), and serum level of cytokine such as IFN-γ, interleukin (IL)-17, and IL-21 were assessed. Peyer's patch cells obtained from mice administered with RG+GABA were cultured, and the cytokine level in the culture supernatant and bone marrow (BM) cell proliferation activity were examined. The proliferative activity of splenocytes was significantly higher in the RG-GABA treatment group than in RG or GABA alone (P < .05). In the experimental tumor metastasis model, oral administration of RG+GABA showed a higher antitumor metastatic effect compared to that of RG or GABA alone. Oral administration of RG+GABA significantly augmented NK cell-mediated cytotoxicity against YAC-1 tumor cells. In addition, the production of GrB and IFN-γ was stimulated in the culture supernatant of NK cells and YAC-1 cells. Serum concentrations of IFN-γ, IL-17, and IL-21 in mice with RG+GABA were significantly higher compared to the corresponding blood levels in mice administered with RG or GABA alone. The RG+GABA group showed significant BM cell proliferation and increased production of IL-6 and granulocyte-macrophage colony-stimulating factor compared to that in the monotherapy groups. Therefore, RG may have a synergistic effect with GABA for enhancing the host defense system such as BM proliferation and NK cell activity in a tumor metastasis model.

Synergistic effect of Korean red ginseng extract and GABA mixture on the IgE production in mice via Th1/Th2 cell balance.

It has been recently reported that the immune system has been linked to the nervous system. This study was conducted to investigate the effect of administration of two components, gamma-aminobutyric acid (GABA) and Panax ginseng Meyer (GIN), on the production of IgE and Th1-Th2 dominant cytokines. Antibody and inflammatory mediator levels in serum, and the cytokines secreted to spleen cells of ovalbumin (OVA) immunized mice were analyzed. The group of GABA and GIN mixture significantly reduced IgE level and dramatically increased OVA-IgG2a antibody production. In addition, rising effect on IFN-gamma and GM-CSF levels related to Th1 cytokine was observed only in the group of GABA + GIN. The mixture alleviated allergic symptoms by reducing the level of histamine and prostaglandin. These studies suggest that GIN + GABA administration in the allergen-induced mouse model may regulate the Th1-Th2 balance by strongly acting on the immune response associated with Th1.

Ring finger protein 219 regulates inflammatory responses by stabilizing sirtuin 1.

BACKGROUND AND PURPOSE: Ring finger protein 219 (RNF219), a protein containing the C3 HC4 -type RING-HC motif, has been identified as a binding partner of the histone deacetylase sirtuin 1 (SIRT1). To explore the functions of RNF219, we examined its possible roles in the cellular responses to inflammation. EXPERIMENTAL APPROACH: Effects of RNF219 on SIRT1 were studied in vitro using RAW264.7 cells and in male BALB/c mice, treated with LPS or IFN-γ. Western blots, RT-PCR, co-immunoprecipitation and ubiquitination assays were used, along with LC-MS/MS analysis. In vivo, survival and serum cytokines and tissue levels of RNF219 and SIRT1 were measured. KEY RESULTS: Binding of RNF219 to SIRT1 inhibited degradation of SIRT1 by preventing its ubiquitination, thereby prolonging SIRT1-mediated anti-inflammatory signalling. LPS caused RNF219 deacetylation, leading to instability of RNF219 and preventing its association with SIRT1. Accordingly, the acetylation status of RNF219 is a critical determinant in its interaction with SIRT1, affecting the response to inflammatory stimuli. The deacetylase inhibitor trichostatin A, increased acetylation and stability of RNF219 and survival of mice injected with LPS, through the interaction of RNF219 with SIRT1. CONCLUSION AND IMPLICATIONS: RNF219 is involved in a novel mechanism to stabilize SIRT1 protein by protein-protein interaction, leading to the resolution of cellular inflammation. These observations provide new insights into the function of RNF219 in modulation of cellular inflammation, and may aid and encourage the development of new anti-inflammatory drugs.

Signaling pathways associated with macrophage-activating polysaccharides purified from fermented barley.

Barley is commonly used in many food and health products. We have previously demonstrated the macrophage-stimulating properties of polysaccharides derived from fermented barley. In this study, three polysaccharide fractions (BF-I-III) were purified from fermented barley and their monosaccharide composition was analyzed. Their immune-stimulatory activities and intracellular signaling pathways were also studied in RAW264.7 cells. Among the three fractions, BF-I exhibited enhanced macrophage activation properties, such as inducing the production of IL-6, IL-12, and TNF-α. However, BF-II and BF-III showed moderate effects on RAW 264.7 cells. BF-I treatment led to the phosphorylation of MAPKs, NF-κB, and c-Jun (major component of AP-1 transcription factor) and induced the nuclear translocation of p65 in RAW264.7 cells. In addition, experiments with neutralizing antibodies showed that Dectin-1, toll-like receptor (TLR) 4, scavenge receptor (SR), and CD14 were mainly involved in the stimulation of nitric oxide (NO) production by BF-I which was suppressed by the inhibition of JNK phosphorylation. These findings suggest that BF-I, isolated from fermented barley, has an immune potentiation activity on macrophages, where it activates the JNK signaling pathway via several macrophage receptors including dectin-1, TLR4, SR, and CD14.

Immunoadjuvant activity in mice of polysaccharides isolated from the leaves of Panax ginseng C.A. Meyer.

Our previous study showed polysaccharide (GS-P) isolated from the leaves of Panax ginseng C.A. Meyer possessed anti-tumor metastatic activity in mouse model. In this study, we evaluated the immunoadjuvant effect of GS-P on the induction of humoral and cellular immune responses against ovalbumin (OVA) in mice. When mice were immunized subcutaneously with OVA admixed with or without GS-P, the OVA+GS-P group showed significantly higher antibody production than the group immunized with OVA alone. This suggests that GS-P has the ability to enhance the adaptive immune response. In addition, the OVA+GS-P+FIA (Freund's incomplete adjuvant) group induced higher levels of antigen-specific IgG1 and IgG2b antibodies than the OVA+FIA group. The culture supernatant obtained from the splenocytes of mice immunized with OVA+GS-P+FIA showed higher levels of OVA-specific Th1-type (IL-2, IFN-γ, GM-CSF) and Th2-type (IL-10) cytokines. Following in vitro analysis of T cell proliferation, the splenocytes of mice treated with OVA+GS-P+FIA showed significantly more proliferation than those treated with OVA+FIA. Further, the production of IgE antibody was dramatically reduced when OVA+GS-P+FIA was used to immunize mice rather than OVA+FIA or OVA+FCA (Freund's complete adjuvant). Collectively, these results suggest that GS-P may possess adjuvant activity that potentially enhances humoral as well as cellular immune responses.

Polysaccharides from ginseng leaves inhibit tumor metastasis via macrophage and NK cell activation.

In this study, we purified the polysaccharide fraction (GS-P) from the leaves of Panax ginseng C.A. Meyer and analyzed its monosaccharide composition and antitumor and antimetastatic activity in vitro and in vivo. GS-P is a 10.2kDa pectic polysaccharide consisting of 15 different monosaccharides. GS-P treatment significantly inhibited metastasis in mice, in a dose-dependent manner. GS-P was not cytotoxic to colon 26-M3.1 cells and increased mouse splenocyte proliferation. Secretion of tumor necrosis factor (TNF)-α and interleukin (IL)-12 was enhanced in the peritoneal exudate macrophages (PEMs) of GS-P-treated mice. Moreover, PEMs obtained from GS-P-treated mice showed significantly higher tumoricidal activity against colon 26-M3.1 cells, and splenocytes from GS-P-treated mice significantly enhanced NK cell cytotoxicity against YAC-1 tumor cells. Pretreatment with anti-asialo GM1 (an antibody for NK cell depletion) partly suppressed the inhibitory effects of GS-P on lung metastasis. These data suggest that GS-P exhibits antimetastatic activity by promoting the activation of macrophages and NK cells.

Changes in the antigenicity and allergenicity of ovalbumin in chicken egg white by N-acetylglucosaminidase.

Ovalbumin (OVA), an (hen) egg allergen, is one of the most abundant glycoprotein allergens associated with IgE-mediated hypersensitivity through the T-helper type 2 immune response. The effect of deglycosylation of the N-terminal glycan in OVA on allergenicity and antigenicity after N-acetylglucosaminidase treatment was studied. N-acetylglucosaminidase-treated OVA (N-OVA) evaluated using an enzyme-linked immunosorbent assay, respectively. N-OVA significantly (p<0.05) OVA-specific IgE and histamine levels. In addition, N-OVA decreased the antigenicity of OVA 1000-fold. These results suggest that the degree of allergenicity and antigenicity reduced with deglycosylation of N-terminal glycan in OVA.

SIGN-R1 and complement factors are involved in the systemic clearance of radiation-induced apoptotic cells in whole-body irradiated mice.

Although SIGN-R1-mediated complement activation pathway has been shown to enhance the systemic clearance of apoptotic cells, the role of SIGN-R1 in the clearance of radiation-induced apoptotic cells has not been characterized and was investigated in this study. Our data indicated that whole-body γ-irradiation of mice increased caspase-3(+) apoptotic lymphocyte numbers in secondary lymphoid organs. Following γ-irradiation, SIGN-R1 and complements (C4 and C3) were simultaneously increased only in the mice spleen tissue among the assessed tissues. In particular, C3 was exclusively activated in the spleen. The delayed clearance of apoptotic cells was markedly prevalent in the spleen and liver of SIGN-R1 KO mice, followed by a significant increase of CD11b(+) cells. These results indicate that SIGN-R1 and complement factors play an important role in the systemic clearance of radiation-induced apoptotic innate immune cells to maintain tissue homeostasis after γ-irradiation.

Cleavage of the terminal N-acetylglucosamine of egg-white ovalbumin N-glycans significantly reduces IgE production and Th2 cytokine secretion.

Ovalbumin (OA) is one of the most abundant of the glycoprotein allergens, and induces a T-helper type 2 immune response that results in an IgE-mediated hypersensitivity. In this study, the terminal carbohydrates of N-glycans from intact OA were cleaved with the exoglycosidases galactosidase, mannosidase, and N-acetylglucosaminidase to generate degalactosylated-OA, demannosylated-OA, and de-N-acetylglucosaminylated-OA, respectively, in order to evaluate their role in allergenicity. The exoglycosidase digestion procedure did not result in either degradation or contamination of the three deglycosylated sample, and the digestion efficiency was confirmed by comparing the results of glycan analysis of the three exoglycosidase-treated OAs with that of glycans of intact OA. Mice were immunized with either intact or exoglycosidase-treated OAs, and their respective allergic reactions were compared. IgE production in the de-N-acetylglucosaminylated-OA group was reduced to 58.8% of that in the intact OA group. In addition, the production levels of the cytokines interleukin-4 and interleukin-5 were significantly reduced in the de-N-acetylglucosaminylated-OA group to 53.4% and 45.8% of the levels in the intact OA group, respectively. However, there were almost no changes (or only slight reductions) in the degalactosylated-OA and demannosylated-OA groups, respectively. These results indicate that cleavage of the terminal carbohydrate, and particularly N-acetylglucosamine, reduces the allergenicity of OA. This is the first report of the effect of cleavage of the terminal carbohydrate on glycoprotein allergenicity.

Porcine Splenic Hydrolysate has Antioxidant Activity in vivo and in vitro.

The antioxidant capacity of porcine splenic hydrolysate (PSH) was studied in vitro and in vivo. Peptide hydrolysates were prepared, using the proteolytic enzyme Alcalase(®). The molecular weights of PSH were 37,666, 10,673, 6,029, and 2,918 g/mol. Rats were fed a 5% (w/v) PSH diet, instead of a casein diet, for 4 wk. The food intake, body weight gain, and liver weight of rats in the PSH group were similar to those in the control (CONT) group. There were no differences in the serum total cholesterol, triglyceride, total protein, or albumin levels between PSH and CONT groups. However, the level of in vivo hepatic lipid peroxidation in PSH group was significantly lower than that in CONT. In vivo hepatic catalase and glutathione peroxidase activities in the PSH group were significantly higher than those in the control group. The in vitro protein digestibility of PSH was lower than that of casein. The in vitro trolox equivalent antioxidant capacity of PSH was significantly higher than that of the peptide hydrolysate from casein. The in vitro radical scavenging activities of PSH were significantly higher than those of the peptide hydrolysate from casein. The present findings suggest that porcine splenic peptides improve the antioxidant status in rats by enhancing hepatic catalase and GSH-Px activities, and indicate a potential mechanism of radical scavenging activity during gastrointestinal passage.

Inhibitory effect of chaga mushroom extract on compound 48/80-induced anaphylactic shock and IgE production in mice.

Chaga mushrooms (Inonotus obliquus) are hypothesised to exhibit general immune-potentiating, anti-inflammatory, and antitumor properties, but their anti-allergic activities are not fully understood. Therefore, this study investigated whether a chaga mushroom extract (C-HE) might have anti-allergic activity. This activity was assessed through the levels of the IgE Ab produced in response to an allergen (OVA). The administration of C-HE prophylactically inhibited the systemic anaphylactic shock induced by compound 48/80 in mice. The oral administration of C-HE significantly reduced the total IgE levels in mice and slightly affected the production of IgG1. Furthermore, spleen cell cultures harvested from OVA-sensitised mice that had received C-HE orally showed a significant increase in Th1-derived responses (IFN-γ production). Therefore, our results suggest that the chaga mushroom extract may be used as an anti-allergic functional food.

The effects of ß-glucans on cancer metastasis.

Beta-glucans (ß-glucans), naturally occurring polysaccharides, are present as constituents of the cell wall of cereal grains, mushrooms, algae, or microbes including bacteria, fungi, and yeast. Since Pillemer et al. first prepared and investigated zymosan in the 1940s and others followed with the investigation of ß-glucans in the 1960s and 1970s, researchers have well established the significant role of ß-glucans on the immune system relative to cancer treatment, infection immunity, and restoration of damaged bone marrow. However, information on their biological role in anti-metastatic activity remains limited. As an immunomodulating agent, ß-glucan acts through the activation of innate immune cells such as macrophages, dendritic cells, granulocytes, and natural killer cells. This activation triggers the responses of adaptive immune cells such as CD4(+) or CD8(+) T cells and B cells, resulting in the inhibition of tumor growth and metastasis. Reports have shown that ß-glucans exert multiple effects on cancer cells and cancer prevention. However the mechanisms of their actions appear complex due to differences in source, chemical structure, insufficiently defined preparation, and molecular weight, hence the inconsistent and often contradictory results obtained. This review is focused on the potential of ß-glucans as anti-metastatic agents and the known mechanisms underlying their biological effects.

Fermentation of Korean red ginseng by Lactobacillus plantarum M-2 and its immunological activities.

We investigated ginsenoside transformation by fermentation of red ginseng with Lactobacillus plantarum M-2. We also examined the anti-metastasis and immune-stimulating activities of EtOH extracts of fermented red ginseng (FRG-E) in animal and human subjects. Total sugar decreased from 85.5 mg mL(-1) to 44.1 mg mL(-1) with increasing culture time during the fermentation with L. plantarum M-2. Uronic acid content reached a maximum level (534.3 µg mL(-1)) at 3 days of fermentation and decreased thereafter. Ginsenoside metabolites increased from 4,637.0 to 7,581.1 µg mL(-1) after 4 days. The prophylactic intraperitoneal injection of FRG-E (500 µg mouse(-1)) inhibited lung metastasis about 81.1%, while the inhibitory effect against tumor metastasis by treatment of EtOH extract from non-fermented red ginseng (NFRG-E) was 66.9%. Immunoglobulin A (IgA) and G (IgG) levels in the serum of healthy subjects were higher after FRG-E administration than at baseline, whereas NFRG-E induced reductions of these variables related to immunity. At 1 week, the change in IgA level by FRG-E (5.14 mg mL(-1)) was significantly higher than that by NFRG-E (-14.50 mg mL(-1); p < 0.05). It was concluded that the immunological activities of FRG-E were higher than those of NFRG-E, indicating that fermentation helped enhance the immunological activities of red ginseng.

Enhancement of anti-complementary and radical scavenging activities in the submerged culture of Cordyceps sinensis by addition of citrus peel.

To investigate the optimal conditions for the production of Cordyceps sinensis by the submerged culture method, glucosamine and exopolysaccharide (EPS) productivities were determined in culture broth containing different carbon sources, principally rice bran and citrus peel. An optimal medium composition (1.5% rice bran, 0.5% molasses, 3% CSL, 0.1% KH(2)PO(4), and 0.05% MgSO(4)) and the optimal condition (25 degrees C and 5-6 d culture time) for high EPS productivity with potent immune-stimulating activities were obtained. The addition of citrus peel to the culture of C. sinensis under the optimized conditions improved EPS productivity and glucosamine content. Furthermore, anti-complementary activity was higher (58.0-80.8%) using citrus peel as compared to no addition of citrus peel (48.2-68.7%). Antioxidant activity (AEAC value) of the citrus peel culture was high (284.3-384.6 mg/100g) compared to that of the culture without citrus peel (142.8-219.5mg/100g), indicating that the citrus peel helped enhance the anti-complementary and antioxidant activities of C. sinensis.

The antioxidant and chemopreventive potentialities of Mosidae (Adenophora remotiflora) leaves.

Our study focused on the antioxidant activities of Mosidae leaf ethanol extract (MLE) and included measurements of reducing power, total phenolic compounds, DPPH radical scavenging activity, and hydroxyl radical scavenging activity. In order to determine whether or not MLE evidences any chemopreventive activities, experimental lung metastasis was induced via the i.v. inoculation of colon26-M3.1 carcinoma cells into BALB/c mice. Additionally, we attempted to characterize any possible cytotoxic effects in murine normal splenocytes and tumor cells (B16-BL6 and colon26-M3.1). The total phenolic content and reducing capacity were measured at 39 mg/100 mL and 1.24, respectively, whereas the DPPH and hydroxyl radical scavenging activities of MLE were measured to be 88.89% and 22.10%, respectively. Prophylactic i.v. treatment with MLE resulted in a dose-dependent and significant inhibition of lung metastasis. Specifically, a MLE dose of 200 ug per mouse resulted in an 88.90% inhibition of lung metastasis. For the cytotoxicity assay, MLE doses up to 100 ug/mL were not shown to affect the growth of normal murine splenocytes. Additionally, the survival of normal cells was not affected at MLE doses below 500 ug/mL. However, MLE doses up to 500 ug/mL reduced the percentage of tumor cell growth for B16BL6 (67% alive) and colon26-M3.1 (62% alive) cells.

Innate immune stimulation of exo-polymers prepared from Cordyceps sinensis by submerged culture.

After we prepared exo-polymers (EPS) from Cordyceps sinensis by submerged culture, prophylactic intravenous administration (i.v.) of EPS significantly inhibited metastasis in experimental lung metastasis of colon 26-M3.1 carcinoma. Cytotoxicity against Yac-1 tumor cells of natural killer (NK) cell, which was prepared by i.v. of EPS (100 mug/mouse), significantly augmented 2 days after EPS treatment. When NK cells were depleted by rabbit anti-asialo GM1 serum, even the EPS group totally abolished the inhibitory effect on lung metastasis of colon 26-M3.1 cells. EPS can stimulate innate immune system to inhibit tumor metastasis, and its anti-tumor metastasis is associated with macrophage and NK cell activation.

Anti-tumor immunostimulatory effect of heat-killed tumor cells.

As a part of our ongoing search for a safe and efficient anti-tumor vaccine, we attempted to determine whether the molecular nature of certain tumor antigens would influence immune responses against tumor cells. As compared with freeze-thawed or formaldehyde-fixed tumor antigens, heat-denatured tumor antigens elicited profound anti-tumor immune responses and greatly inhibited the growth of live tumor cells. The heat-denatured tumor antigens induced a substantial increase in the anti-tumor CTL response in the absence of any adjuvant material. This response appears to be initiated by strong activation of the antigen-presenting cells, which may recognize heat-denatured protein antigens. Upon recognition of the heat-denatured tumor antigens, macrophages and dendritic cells were found to acutely upregulate the expression of co-stimulatory molecules such as B7.2, as well as the secretion of inflammatory cytokines such as IL-12 and TNF-alpha. The results of this study indicate that heat-denatured tumor extracts might elicit protective anti-tumor adaptive immune responses and also raise the possibility that a safe and efficient adjuvant-free tumor vaccine might be developed in conjunction with a dendritic cell-based tumor vaccine.

Anti-tumor metastatic activity of beta-glucan purified from mutated Saccharomyces cerevisiae.

The beta-glucans isolated from Saccharomyces cerevisiae (S. cerevisiae) enhance the innate immune system, but there is little evidence for its antitumor activity. To examine the antitumor and immunostimulating activities of beta-glucan (IS-2) purified from mutated S. cerevisiae, we made an experiment on innate immune response against metastasis of cancer cells by comparing with the beta-glucan from wild-type S. cerevisiae. In experimental lung metastasis of colon 26-M3.1 carcinoma or B16-BL6 melanoma cells, prophylactic administration of beta-glucan purified from mutated S. cerevisiae significantly inhibited lung metastasis in a dose-dependent manner. Furthermore, therapeutic administration of IS-2 also significantly inhibited the colon 26-M3.1 cell growth in mice. In an assay of liver and spleen metastasis produced by i.v. inoculation of L5178Y-ML25 lymphoma cells, IS-2 also significantly inhibited metastasis in CDF1 mice. Furthermore, pretreatment with IS-2 two days before tumor inoculation significantly prolonged the survival time of tumor-bearing mice. In an in vitro cytotoxicity analysis, IS-2 (up to 100 microg/ml) did not affect the growth of colon 26-M3.1 cells. In contrast, IS-2 enhanced splenocyte proliferating activity in a dose-dependent manner. Peritoneal macrophages stimulated with IS-2 produced various cytokines, such as IL-1beta, IFN-gamma, and IL-12. In addition, treatment with IS-2 (20 microg/mouse) induced tumoricidal activity of peritoneal macrophages against colon 26-M3.1 cells. In an assay for natural killer (NK) cell activity, IS-2 (20 microg/mouse, i.v.) significantly augmented NK cytotoxicity against Yac-1 tumor cells at 2 days after IS-2 treatment. The depletion of NK cells by injection of rabbit anti-asialo GM1 serum abolished the inhibitory effect of IS-2 on lung metastasis of colon 26-M3.1 cells. These data suggest that IS-2 inhibits tumor metastasis via activation of macrophages and NK cells.

Dietary Korean mistletoe enhances cellular non-specific immune responses and survival of Japanese eel (Anguilla japonica).

The present study was performed to investigate the immunostimulatory effects of Korean mistletoe extract (KM-110; Viscum album Coloratum) on the non-specific immune response and protection against Aeromonas hydrophila infection in Japanese eel (Anguilla japonica). Eels were fed under 4 regimes, 0%, 0.1%, 0.5% and 1.0% KM-110 mixed diet. On day 14 after feeding, 15 fish from each group were injected i.p. with live A. hydrophila (3 x 10(6)CFU) and the remaining unchallenged fish from each group were used to study the innate immune response. On 14 days post-infection, the total survival rates were 26.6% in control, and 33.3%, 66.6% and 80% in 0.1%, 0.5% and 1% KM-110-treated groups, respectively. The maximum lysozyme activity was observed in the 1% KM-110-treated group. There was no significant difference of lysozyme activity between 0.1% and 0.5% KM-110 group. Superoxide anion (O(2)(-)) production was significantly (p<0.05) augmented in the 0.5% and 1% KM-110 groups compared to the control and 0.1% KM-110 group. No significant difference of (O(2)(-) production was found between 0.5% and 1% KM-110 group. Likewise, there was a significant increase in phagocytic activity in the 0.5% KM-110 group compared with the 0.1% group (p<0.05), but no significant difference between the 0.5% and the 1% KM-110 group indicating that 0.5% KM-110 concentration is suitable for stimulating maximum phagocytic activity resulting in a high amount of ROI production. Considering the present results, KM-110 could be utilized as a promising immunostimulating substance for a diet in aquaculture.