A 75 kDa glycoprotein isolated from Cudrania tricuspidata Bureau induces colonic epithelial proliferation and ameliorates mouse colitis induced by dextran sulfate sodium.

Cudrania tricuspidata Bureau (CTB), a species of the Moraceae plant, has been used as a bruise recovery treatment. This study aimed to determine whether the 75 kDa phytoglycoprotein extracted from CTB has a regulatory effect on the proliferation of human colon epithelial cells and the pathological process of inflammatory bowel disease (IBD). We found that CTB glycoprotein significantly induces the proliferation of human colon epithelial HT-29 cells by activating protein kinase C. CTB glycoprotein stimulated the phosphorylation of c-Jun N-terminal kinase and transcription factor nuclear factor-κB, which are responsible for the expression of cell-cycle-related proteins (CDK2, CDK4, cyclin D1 and cyclin E) during its promotion of cell proliferation. Experimental colitis was induced in mice by adding dextran sulfate sodium to their drinking water at a concentration of 4% (W/V) for seven days. We found that CTB glycoprotein ameliorates the pathological process of IBD and lowers the disease activity index score, which was composed of body weight change, diarrhea, and hematochezia in ICR mice treated with dextran sulfate sodium. Hence, we suggest that CTB glycoprotein has the ability to prevent IBD by promoting cell proliferation signaling events via the activation of PKC, JNK and NF-κB in colon epithelial cells.

Phytoglycoprotein isolated from Dioscorea batatas Decne promotes intestinal epithelial wound healing.

Dioscorea batatas Decne (DBD) has been used to heal various illnesses of the kidney and intestine as an herbal medicine in Asia. As a source of therapeutic agents, many glycoproteins have been isolated from mushrooms and plants, but the functional role of glycoprotein in intestinal epithelial wound healing has not been understood yet. In the present study, we investigated the wound healing potentials of the 30 kDa glycoprotein (DBD glycoprotein) isolated from DBD in human intestinal epithelial (INT-407) cells. We found that DBD glycoprotein (100 µg·mL-1) significantly increased the motility of INT-407 cells for 24 h by activating protein kinase C (PKC). DBD glycoprotein stimulated the activation of p38 mitogen-activated protein kinase (MAPK), which is responsible for the phosphorylation of NF-κB inhibitor α (IκBα). DBD glycoprotein increased the level of profilin-1 (PFN1), α-actinin and F-actin expression via activation of transcription factor, nuclear factor-kappa B (NF-κB) during its promotion of cell migration. Experimental mouse colitis was induced by adding dextran sulfate sodium (DSS) to the drinking water at a concentration of 4% (W/V) for 7 days. We figured out that administration of DBD glycoprotein (10 and 20 mg·kg-1) lowers the levels of disease activity index and histological inflammation in DSS-treated ICR mice. In this regard, we suggest that DBD glycoprotein has ability to promote the F-actin-related migration signaling events via activation of PKC and NF-κB in intestinal epithelial cells and prevent inflammatory bowel disease.

Intestinal epithelial cell apoptosis due to a hemolytic toxin from Vibrio vulnificus and protection by a 36 kDa glycoprotein from Rhus verniciflua Stokes.

Rhus verniciflua stokes (RVS) has been used as a functional food to cure inflammatory diseases in Korea. In the present study, we carry out an investigation of the cellular mechanism of a 36 kDa glycoprotein isolated from RVS fruit (RVS glycoprotein) during the apoptosis of human gastrointestinal epithelial HCT116 cells induced by the hemolytic toxin (VvhA) produced by V. vulnificus. Recombinant protein (r) VvhA produced by V. vulnificus stimulated apoptosis by activating the phosphorylation of protein kinase C (PKC) through the production of intracellular reactive oxygen species (ROS). However, RVS glycoprotein significantly inhibited the level of ROS production and PKC activation in rVvhA-stimulated HCT116 cells. Interestingly, we found that RVS glycoprotein has inhibitory effects on the phosphorylation of c-Jun N-terminal kinase (JNK) and nuclear factor-kappa B (NF-κB), which are responsible for the expression of Bax and cleaved caspase-3 in HCT116 cells treated with rVvhA, respectively. On the basis of these results, we suggest that RVS glycoprotein blocks mitochondrial apoptotic cell death induced by rVvhA via the inhibition of ROS-mediated signaling events in HCT116 cells.

The anti-allergic activity of Lactobacillus plantarum L67 and its application to yogurt.

Recently, interest in the beneficial role of probiotics in the protection and management of allergic diseases caused by immune disorders has been increasing. This study investigated the inhibitory effect of Lactobacillus plantarum L67 on induced allergic inflammatory response in bisphenol A-treated rat basophilic leukemia 2H3 (RBL-2H3) cells and mouse splenocytes. We also evaluated the applicability of L. plantarum L67 as a yogurt starter culture. We measured the ability of Lactobacillus strains to induce the production of IL-12 and IFN- γ in cultured splenocytes by ELISA. Bisphenol A (50µM)-treated RBL-2H3 cells were cotreated with a glycoprotein (18kDa) isolated from L. plantarum L67 (5-100µg/mL) for 30min. We measured the expression of mitogen-activated protein kinase (ERK and p38), AP-1 (c-Fos and c-Jun), T-bet, and GATA-binding protein 3 (GATA-3) using Western blotting to examine the differentiation of T helper cells. Furthermore, we evaluated the gene expression of IL-1ß, IL-6, and IL-10 using real-time quantitative PCR. Finally, we evaluated the applicability of L. plantarum L67 as a yogurt starter by measuring pH, enumeration of bacteria, and sensory scores. Our results showed that L67 protein inhibited the phosphorylation of ERK and p38 mitogen-activated protein kinase through the transcriptional activation of AP-1 in bisphenol A-treated RBL-2H3 cells. During differentiation of T helper cells, the expression of transcription factor GATA-3 was significantly suppressed by L67 protein (100µg/mL) treatment, whereas expression of transcription factor T-bet was increased. In addition, the L67 protein significantly attenuated the expression of T helper 2-linked cytokines IL-1ß, IL-6, and IL-10. These results indicate that L. plantarum L67, made available as yogurt starters and dietary supplements, has the potential to prevent allergy-related immune disorders.

Lactobacillus plantarum L67 glycoprotein protects against cadmium chloride toxicity in RAW 264.7 cells.

The food and water we consume may be contaminated with a range of chemicals and heavy metals, such as lead, cadmium, arsenic, chromium, and mercury by accumulation through the food chain. Cadmium is known to be one of the major components in cigarette smoke and can cause lesions in many organs. Some lactobacilli can bind and remove heavy metals such as cadmium, lead, and copper. However, the mechanisms of cadmium toxicity and inhibition by probiotics are not clear. In this study, we demonstrated that glycoprotein (18 kDa) isolated from Lactobacillus plantarum L67 protected RAW 264.7 cells from expression of inflammation-related factors stimulated by cadmium chloride (100 µM). Furthermore, we evaluated the cytotoxicity of cadmium using the MTT assay and intracellular Ca(2+) using fluorescence, and assessed activities of activator protein kinase C (PKC-α), inducible nitric oxide synthase, activator protein (AP)-1, and mitogen-activated protein kinases using immunoblot. Our results indicated that glycoprotein isolated from L. plantarum L67 inhibited intracellular Ca(2+) mobilization. It also significantly suppressed inflammatory factors such as AP-1 (c-Jun and c-Fos), mitogen-activated protein kinases (ERK, JNK, and p38), and inducible nitric oxide synthase. Our findings suggest that the 24-kDa glycoprotein isolated from L. plantarum L67 might be used as a food component for protection of inflammation caused by cadmium ion.

Glycoprotein isolated from Styrax japonica Siebold et al. Zuccarini inhibits oxidative and pro-inflammatory responses in HCT116 colonic epithelial cells and dextran sulfate sodium-treated ICR mice.

This study was carried out to investigate the anti-inflammatory potentials of a 38 kDa glycoprotein isolated from Styrax japonica Siebold et al Zuccarini (SJSZ glycoprotein). We found that SJSZ glycoprotein has concentration-dependent scavenging activity against DPPH and hydroxyl radicals in the cell-free systems. In colonic epithelial cells (HCT116 cells), the results showed that SJSZ glycoprotein inhibits the production of reactive oxygen species (ROS) induced by glucose/glucose oxidase (G/GO) in a concentration-dependent manner. Experimental mouse colitis was induced by adding dextran sulfate sodium (DSS) to the drinking water at a concentration of 4% (w/v) for 7 days. We figured out that administration of SJSZ glycoprotein (10 mg/kg) lowers the levels of disease activity index, myeloperoxidase activity, and histological inflammation in DSS-treated mice. In addition, SJSZ glycoprotein inhibited plasmic thiobarbituric acid reactive substances (TBARS) formation, nitric oxide (NO) production, and lactate dehydrogenase (LDH) release, accompanying the inhibition of colonic inflammatory signal proteins (NF-κB, iNOS, and COX-2) and inflammation-related cytokines (IL-1ß, IL-6, and TNF-α). These results indicate that SJSZ glycoprotein inhibits oxidative and pro-inflammatory responses in mouse colitis.

Preventive effects of ZPDC glycoprotein (24 kDa) on hepatotoxicity induced by mercury chloride in vitro and in vivo.

Mercury is a potent environmental contaminant that exerts toxic effect on various vital organs in the human body. Recently, we isolated glycoprotein from Zanthoxylum piperitum DC (ZPDC), which has antioxidant and anticancer effects. In the present study, we determined the preventive effects of ZPDC glycoprotein on hepatic damage induced by mercury chloride (HgCl2 ). We evaluated the activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)], extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), cyclo-oxygenase (COX-2), inducible nitric oxide synthetase (iNOS), and activator protein (AP-1) and the quantitative expressions of nuclear factor E2-related factor (Nrf2), heme oxygenase (HO-1), metallothionein (MT) and reduced glutathione (GSH) in mercury-chloride-exposed (50 µM and 10 mg/kg body weight) primary cultured hepatocytes and ICR mice, using biochemical assays, radioactivity and immunoblot analysis. The results demonstrated that ZPDC glycoprotein decreased the levels of LDH, ALT, HO-1 and MT, whereas it increased the activities of hepatic antioxidant enzymes (SOD, CAT and GPx) and reduced GSH in mercury-chloride-exposed primary cultured hepatocytes. Also, it suppressed arachidonic acid release and expression of ERK, p38 MAPK, COX-2, iNOS, AP-1 and Nrf-2 in primary cultured hepatocytes and ICR mice exposed to mercury chloride. Collectively, ZPDC glycoprotein may have potential applications to prevent hepatotoxicity induced by mercury chloride.

ZPDC glycoprotein (24 kDa) induces apoptosis and enhances activity of NK cells in N-nitrosodiethylamine-injected Balb/c.

Natural killer (NK) cells have anti-tumor activity in hepatocellular carcinoma (HCC) using secreting granules and cytotoxic ability. Recently, we isolated glycoprotein from Zanthoxylum piperitum DC (ZPDC) has anti-oxidant effect and anti-cancer effect. The objective of this study was to determine whether ZPDC glycoprotein enhances activity of NK cells and induces apoptosis of liver cancer cells in diethylnitrosamine (DEN)-treated Balb/c mice. This study evaluated the secreting of perforin and granzyme B and cytotoxicity of NK cells, interleukin (IL)-2 and IL-12, apoptosis-related factors (bid, cytochrome c, and caspase-3) in liver tissue using Immunoblot and ELISA. The results demonstrated that ZPDC glycoprotein (20mg/kg, BW) induces secretion of perforin and granzyme B and NK cells activity. Also, it induces expression of apoptosis-related factors (bid, cytochrome c, and caspase-3) in liver tissues. Collectively, ZPDC glycoprotein may have potential applications to prevent hepatocarcinogenesis without immunosuppression.

SJSZ glycoprotein (38 kDa) modulates macrophage type 1/2-related factors at hepatocarcinogenic stage in N-nitrosodiethylamine-treated Balb/c.

Macrophage plays critical role for tumor progression: Type 1 (M1) for tumor prevention and type 2 (M2) for promotion in hepatocellular carcinoma. In order to study the chemopreventive effects of the SJSZ glycoprotein (38 kDa) on M1- or M2-related factors, Balb/c was injected intraperitoneally with N-nitrosodiethylamine (DEN; 50 mg/kg, BW) for 7 weeks. After 7 weeks, the mice were sacrificed. After that, peritoneal macrophages were isolated. We evaluated the production of reactive oxygen species (ROS) and nitric oxide (NO), hepatocarcinogenic signals [activities of mitogen-activated associated kinase (MAPKs), inducible nitric oxide synthase (iNOS), nuclear factor (NF)-κB, and signal transducer and activator of transcription (STAT) 6,], cytokines [interleukin (IL)-10, IL-4, IL-12, and interferon (IFN)-γ], and CD163-positive macrophages (M2 polarization) using biochemical methods, immunoblot analysis, qRT-PCR, ELISA, and flow cytometry. The results revealed that the SJSZ glycoprotein (10 mg/kg, BW) inhibits the phosphorylation of MAPKs and expression of NF-κB, pSTAT6, IL-10, and IL-4; and normalizes production of ROS and NO, and expression of iNOS, IL-12, and IFN-γ. Especially, it inhibited CD163-positive macrophages. In conclusion, these results indicated that SJSZ glycoprotein modulates polarization of macrophage type 1 and type 2 at hepatocarcinogenic initial stage in DEN-treated Balb/c. Thus, SJSZ glycoprotein may be useful as one of immunomodulating agents which have to regulate M1- and M2-related factors to prevent tumor progression.

Inductive effect of phytoglycoprotein (38 kDa) on G0/G1 arrest and apoptosis in diethylnitrosamine-treated ICR mice.

Promotion in the carcinogenic stage is closely related to the growth and proliferation of liver cancer cells. The purpose of this study was whether Styrax japonicum Siebold & Zuccarini (SJSZ) glycoprotein has preventive effect on the growth of hepatocarcinoma cells in diethylnitrosamine (DEN)-induced ICR mice. The study evaluated cell cycle and the activities of cell cycle-related factors [cyclin D1/cyclin dependent kinase (CDK) 4], cell cycle inhibitors (CKIs; p53, p21, and p27), proliferating cell nuclear antigen (PCNA), cytochrome c, Bid, caspase-3, and caspase-9 in DEN-induced ICR mice by flow cytometry, immunoblot analysis and qRT-PCR. The results showed that SJSZ glycoprotein (10 mg/kg, BW) arrested G(0)/G(1) phase and the activity of cyclin D1/CDK4, PCNA, and Bid. However, it induced activities of CKIs, cytochrome c, caspase-3, and caspase-9. Taken together, this present study suggested that SJSZ glycoprotein might be a potent inhibition of hepatic tumor promotion.

Growth of HepG2 cells was suppressed through modulation of STAT6/IL-4 and IL-10 in RAW 264.7 cells treated by phytoglycoprotein (38 kDa).

Macrophage type 2 (M2) is closely associated with tumor progression and metastasis. Thus, in this study, the antitumor effect of Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein on HepG2 cell proliferation through modulating M2 was investigated by measuring [³H]-thymidine incorporation and proliferating cell nuclear antigen (PCNA), nitric oxide (NO), reactive oxygen species (ROS), mitogen-activated protein kinases, signal transducer and activator of transcription (STAT) 6, cytokines [interleukin (IL)-4, IL-10, IL-12, and interferon (IFN)-γ], and CD163-positive cells using biochemical analysis, radioactivity, Western blot, ELISA, quantitative real-time polymerase chain reaction, and flow cytometry in coculture system. RAW 264.7 cells were found to be cytotoxic to HepG2 cells but [³H]-thymidine incorporation and expression of PCNA was suppressed in the presence of the SJSZ glycoprotein (20 µg/ml). The SJSZ glycoprotein normalized production of NO and ROS and expression of inducible nitric oxide synthase, IFN-γ, and IL-12 but suppressed expression of pSTAT6, IL-4, IL-10, and CD163-positive cells. Thus, the results of this study suggest that the SJSZ glycoprotein suppresses proliferation of HepG2 cells by modulating M2.

SJSZ glycoprotein (38 kDa) inhibits cell cycle and oxidative stress in N-methyl-N'-nitro-N-nitrosoguanidine-induced ICR mice.

The initiation stage of liver cancer is closely related to abnormal cell proliferation as observed for other types of carcinogenesis. Recently, we isolated a glycoprotein from Styrax japonica Siebold et al Zuccarini (SJSZ glycoprotein), which consists of a carbohydrate moiety (52.64%) and a protein moiety (47.36%). In this study, the antitumoric mechanism of SJSZ glycoprotein during the initiation stage in N-Methyl-N`-nitro-N-nitrosoguanidine (MNNG; 40 mg/kg, BW)-induced ICR was investigated. First, we evaluated the activities of lactate dehydrogenase (LDH), alanine aminotransferase (ALT), thiobarbituric acid-reactive substances (TBARS), and activities of antioxidative enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)] in mouse liver tissue and serum. The alpha-fetoprotein (AFP), cell cycle-related factors [cyclin D1/ cyclin dependent kinase (CDK) 4], cell cycle inhibitors (CKIs; p53, p21, and p27), and proliferating cell nuclear antigen (PCNA) were then assessed using Western Blot analysis. The results of this analysis showed that the SJSZ glycoprotein (10 mg/kg, BW) decreased the levels of LDH, ALT, TBARS, and the expression of AFP but it increased the activity of hepatic anti-oxidant enzymes (SOD, GPx and CAT). In addition, the SJSZ glycoprotein (10 mg/kg, BW)was shown to decrease the expression of cyclin D1/CDK4 and PCNA and increase the expression of CKIs (p53, p21, and p27). The results in this study indicate that the SJSZ glycoprotein displays anti-oxidative stress and anti-cell proliferation activity in MNNG induced ICR.

Normalizing effect of SJSZ glycoprotein (38 kDa) on proliferating cell nuclear antigen and interferon-γ in diethylnitrosamine-induced mice splenocytes.

One of the immunosuppressive responses when hepatocellular carcinoma (HCC) develops in mammals is defective proliferation in the spleen. The objective of this study was to investigate the protective effect of the Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein on the proliferation of splenocytes induced by diethlynitrosamine (DEN). To assess whether the SJSZ glycoprotein modulates splenocyte proliferation, Balb/c mice were injected intraperitoneally with DEN (50 mg/kg, BW) for 7 weeks. After 7 weeks, the mice were sacrificed, and spleens were isolated. We evaluated [(3) H]-thymidine incorporation, extracellular signal-regulated kinase (ERK), cell cycle-related factors [p53, p21, p27, cyclin D1/cyclin dependent kinase (CDK) 4], proliferating cell nuclear antigen and interferon (IFN)-γ using radiation activity, immunoblot analysis, and the reverse transcription-polymerase chain reaction. The results revealed that the SJSZ glycoprotein (10 mg/kg, BW) increased [(3) H]-thymidine incorporation, ERK phosphorylation, expression levels of cyclin D1/cyclin dependent kinase 4, and IFN-γ. However, the SJSZ glycoprotein decreased levels of p53, p21, and p27. Taken together, these results suggest that the SJSZ glycoprotein inhibited defective splenocyte proliferation induced by DEN.

Protection against cyclophosphamide-induced myelosuppression by ZPDC glycoprotein (24 kDa).

Immunomodulatory agents are often used to reduce myelosuppression and enhance immune response for cancer treatment. Cyclophosphamide (CTX) can induce oxidative stress in bone marrow resulting in suppression of anti-oxdiantive enzymes and causes myelosuppression. We isolated glycoprotein from Zanthoxylum piperitum DC fruit (ZPDC), and it consists of a carbohydrate (18%) and a protein (82%). The objective of this study was to investigate its protective activity against CTX-induced myelosuppression in Balb/c (n=6/group). The mice were orally administrated by ZPDC glycoprotein (10 and 20 mg/kg, BW) for 1 week in the presence or absence of CTX. Intracellular reactive oxygen species (ROS), anti-oxidant enzymes [superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT)], cyclin kinase inhibitors (CKIs: p53, p21 and p27), cyclin D1/ cyclin dependent kinase (CDK) 4, PCNA and cytokines [interleukin (IL)-3, and granulocyte/ macrophage-colony-stimulating factor (GM-CSF)] were evaluated using biochemical activity, Western blot analysis, and ELISA. The results obtained from this study showed that CTX decreased spleen and thymic indices, bone marrow cellularity and expression of cyclin D1/CDK4 and PCNA, but it increased CKIs, whereas ZPDC glycoprotein (20 mg/kg, BW) resulted in vice versa in CTX-induced Balb/c. Expression of IL-3 and GM-CSF were normalized by ZPDC glycoprotein. Thus, this study suggested that ZPDC glycoprotein prevents oxidative stress and myelosuppression in CTX-induced mice and might be a potential immunomodulatory agent.

SJSZ glycoprotein (38 kDa) modulates expression of IL-2, IL-12, and IFN-γ in cyclophosphamide-induced Balb/c.

OBJECTIVE: Cyclophosphamide (CTX) often results in immunosuppression and cytotoxic effects. The object of this study was to understand whether Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein prevents immunosuppression in CTX-induced Balb/c. METHODS: The mice were injected intraperitoneally with CTX (80 mg/kg, BW) for 1 week in the presence or absence of the SJSZ glycoprotein, and divided into five groups. Weights of the spleen and thymus, phagocytic macrophages, proliferation of splenocytes and thymocytes ([(3)H]-thymidine incorporation and expression of PCNA), natural killer (NK) cytotoxicity [MTT assay, perforin, and granzyme B], and cytokines [interleukin (IL)-2, IL-12, and interferon (IFN)-γ] were evaluated using radioactivity, biochemical reactions, immunoblot analysis, and qRT-PCR. Activity of antioxidant enzymes [superoxide dismutase (SOD), glutathione dismutase (GPx) and catalase (CAT)] was also assessed. RESULTS: The results revealed that while the parameters assessed decreased with treatment with CTX alone, SJSZ glycoprotein (10 mg/kg, BW) in the presence of CTX significantly normalized the weights of spleen and thymus, the phagocytic effect of peritoneal macrophages, the activity of antioxidant enzymes, proliferation (splenocytes and thymocytes), NK cell cytotoxicity, and expression of IL-2, IL-12, and IFN-γ. CONCLUSION: SJSZ glycoprotein can normalize activity of anti-oxidative enzymes and immune-related factors.

SJSZ glycoprotein (38kDa) prevents thymus atrophy and enhances expression of IL-2 and IL-12 in diethylnitrosamine-induced hepatocarcinogenesis.

Hepatocellular carcinoma (HCC) is a typical inflammation-associated cancer, but has also been shown to provoke antitumor immune responses. Polarized T helper type 2 (Th2) responses down-regulate antitumor immunity to link with HCC. The objective of this study was to investigate the protective effect of the Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein on thymus atrophy and differential response of Th1/Th2 cells induced by diethlynitrosamine (DEN). To evaluate the modulatory effect of the SJSZ glycoprotein on thymic atrophy and imbalanced Th1/Th2 cells, we examined the weight of the thymus, [(3)H]-thymidine incorporation and expression of proliferating cell nuclear antigen (PCNA), and activities of protein kinase C (PKC)/intracellular Ca(2+), extracellular signal-regulated kinase (ERK)1/2, p38 MAPK, T-box transcription factor (T-bet), GATA-binding protein-3 (GATA-3), cytokines [interleukin (IL)-4, -10, -2, -12 and interferon (IFN)-γ] using radioactivity, immunoblot analysis, and qRT-PCR. The SJSZ glycoprotein (10mg/kg, BW) was shown to increase the weight of the thymus, [(3)H]-thymidine incorporation and PCNA in thymocytes induced by DEN. Also, it increased expression levels of T-bet and Th1 cytokines (IFN-γ, IL-2 and IL-12). However, the activity of PKC/intracellular Ca(2+), phosphorylation of ERK and p38 MAPK, expression levels of GATA-3 and Th2 cytokines (IL-4 and IL-10) were decreased. Taken together, these results suggest that the SJSZ glycoprotein can prevent thymic atrophy and Th2 cytokines induced by DEN.

Inhibitory effect of Styrax Japonica Siebold et al. Zuccarini glycoprotein (38 kDa) on interleukin-1ß and induction proteins in chromium(VI)-treated BNL CL.2 cells.

Chromium(VI) [Cr(VI)] induces chronic inflammation in hepatocytes. Inflammation has been shown to play an important role in tumorigenesis, tumor progression, and metastasis. To examine the effects of the Styrax Japonica Siebold et al. Zuccarini (SJSZ) glycoprotein on inflammation in BNL CL.2 cells, we evaluated the activities of c-Jun NH(2)-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), nuclear factor (NF)-κB (p50 and p65), and inflammation-related factors [cyclooxygenase (COX)-2, inducible nitric oxide syntheses (iNOS) and interleukin (IL)-1ß] in Cr-induced BNL CL.2 cells using immunoblot analysis and RT-PCR. We also used two-dimensional gel electrophoresis (2-DE) to compare between treatments. To determine which proteins are induced by Cr(VI), we evaluated total protein lysates using 2-DE. After Cr(VI) treatment, total protein lysates were prepared and resolved by 2-DE. The results obtained from this study demonstrated that the SJSZ glycoprotein (50 µg/ml) inhibits expression of JNK, ERK, NF-κB, and the expression of COX-2, iNOS, and IL-1ß. Moreover, the results obtained from 2DE showed that four proteins out of nine proteins were relatively expressed strongly, while the rest of them were relatively appeared weakly on the gel. Taken together, these data indicate that the SJSZ glycoprotein prevents expression of COX-2, iNOS, and IL-1ß by blocking NF-κB and MAPKs in Cr(VI)-induced BNL CL.2 cells.

CTB glycoprotein (75kDa) inhibits IgE releasing, TNF-α and IL-6 expressed by bisphenol A in vivo and in vitro.

Cudrania tricuspidata Bureau (CTB) has been used to treat allergies and inflammatory disease as folk medicine in Korea. The objective of this study is to determine whether a glycoprotein isolated from CTB (75 kDa) has a preventive potential of allergic inflammation caused by bisphenol A (BPA) in BALB/c mice and RBL-2H3 cells. Production of immunoglobulin (Ig) E and releasing of ß-hexosaminidase and histamine at treatment of CTB glycoprotein (5-10mg/kg, BW) were evaluated in mice serum. Activation of extracellular signal-regulated kinases (ERK) and p38 mitogen-activated protein kinase (MAPK), activator protein (AP)-1, expressions of pro-inflammatory cytokines, nitric oxide (NO) production and cyclooxygenase (COX)-2 were assessed in RBL-2H3 cells. In the results, CTB glycoprotein (10mg/kg, BW) inhibited the production of IgE and releasing of ß-hexosaminidase and histamine. Also, the CTB glycoprotein (100 µg/ml) blocked phosphorylation of ERK1/2 and p38 MAPK, and the activation of AP-1, while it inhibited the NO production, activities of COX-2, tumor necrosis factor (TNF)-α, and interleukin (IL)-6, but not IL-1ß. Taken together, the results of this study indicated that the CTB glycoprotein modulates the expression of allergic inflammation-related factors via the suppression of MAPK/AP-1 activation.

Inhibitory effect of SJSZ glycoprotein (38 kDa) on expression of heat shock protein 27 and 70 in chromium (VI)-treated hepatocytes.

Chromium (VI) is as an extremely toxic chemical substance, and is also an internationally recognized human carcinogen. The principal objective of this study was to determine whether or not Styrax japonica Siebold et al. Zuccarini (SJSZ) glycoprotein prevents hepatocarcinogenesis in chromium-treated BNL CL.2 cells and ICR mice. Firstly, it was evaluated that SJSZ glycoprotein has strong antioxidant character and scavenges radicals. In an effort to assess the chemopreventive effects of SJSZ glycoprotein on hepatocarcinogenesis, ICR mice were intraperitoneally injected with chromium (10 mg/kg, BW) for 8 weeks. After sacrifice, we evaluated indicators of liver tissue damage [the activities of lactate dehydrogenase (LDH) and alanine aminotransferase (ALT), and thiobarbituric acid-reactive substances (TBARS)], antioxidative enzymes [activities of superoxide dismutase (SOD), catalase (CAT) and gluthathione peroxidase (GPx)], and initiating hepatocarcinogenic indicator [heat shock protein (HSP) 27 and 70] and protein kinase C (PKC), p38 MAPK and PCNA via biochemical methods and immunoblot analysis. The results obtained from this study demonstrated that the SJSZ glycoprotein (50 µg/ml) inhibited the production of intracellular ROS in BNL CL.2 cells. In addition, the SJSZ glycoprotein (10 mg/kg, BW) attenuated the levels of LDH, ALT, and TBARS, whereas it increased antioxidative enzymes in mouse serum. SJSZ glycoprotein attenuated the activity of HSP27, HSP70, PKC, MAPKs, and PCNA in BNL CL.2 cells and liver tissue. Taken together, our results indicate that SJSZ glycoprotein might be have a potent preventive effect against hepatocarcinogenesis induced by oxidative stress.

Modulatory effect of phytoglycoprotein (38 kDa) on cyclin D1/CDK4 in BNL CL.2 cells induced by N-methyl-N'-nitro-N-nitrosoguanidine.

In the developmental stages of cancer, cell transformation occurs after the promotion stage and is a marker of cancer progression. This cell transformation is related to abnormal proliferation during the cancer initiation stage. The purpose of this study was to evaluate the effect of Styrax japonica Siebold et al. Zuccarin (SJSZ) glycoprotein on cell transformation in murine embryonic liver cells (BNL CL.2) following N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment. To determine abnormal proliferation during the initiation stage, intracellular reactive oxygen species (ROS), phosphorylation of extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), activities of cell cycle-related factors [cyclin D1/cyclin dependent kinase (CDK) 4], cell cycle inhibitors (p53, p21, and p27), nuclear factor (NF)-κB, and proliferating cell nuclear antigen (PCNA) were evaluated using Western blot analysis and real-time PCR. Our study demonstrated that SJSZ glycoprotein (50 µg/ml) reduces foci formation with combined treatment [MNNG and 12-O-tetradecanoyl phorbol-13-acetate] of BNL CL.2 cells. With regard to proliferation-related signals, our finding indicated that SJSZ glycoprotein (50 µg/ml) diminished the production of intracellular ROS, activity of phosphorylated ERK, p38 MAPK, NF-κB (p50 and p65), PCNA, and cyclin D1/CDK4 in MNNG-induced BNL CL.2 cells. Taken together, these results lead us to speculate that SJSZ glycoprotein can inhibit abnormal cell proliferation at the initiation stage of hepatocarcinogenesis.