Euikyung Kim

Fucoidan from seaweed Fucus vesiculosus inhibits migration and invasion of human lung cancer cell via PI3K-Akt-mTOR pathways.

Background Recently there has been an increased interest in the pharmacologically active natural products associated with remedies of various kinds of diseases, including cancer. Fucoidan is a polysaccharide derived from brown seaweeds and has long been used as an ingredient in some dietary supplement products. Although fucoidan has been known to have anti-cancer activity, the anti-metastatic effects and its detailed mechanism of actions have been poorly understood. Therefore, the aims of this study were to demonstrate the anti-metastatic functions of fucoidan and its mechanism of action using A549, a highly metastatic human lung cancer cell line. Methods and Principal Findings Fucoidan inhibits the growth of A549 cells at the concentration of 400 µg/ml. Fucoidan treatment of non-toxic dose (0–200 µg/ml) exhibits a concentration-dependent inhibitory effect on the invasion and migration of the cancer cell via decreasing its MMP-2 activity. To know the mechanism of these inhibitory effects, Western blotting was performed. Fucoidan treatment down-regulates extracellular signal-related kinase 1 and 2 (ERK1/2) and phosphoinositide 3-kinase (PI3K)–Akt–mammalian target of rapamycin (PI3K-Akt-mTOR) pathways. Furthermore, fucoidan decreases the cytosolic and nuclear levels of Nuclear Factor-kappa B (p65). Conclusions/Significance The present study suggests that fucoidan exhibits anti-metastatic effect on A549 lung cancer cells via the down-regulation of ERK1/2 and Akt-mTOR as well as NF-kB signaling pathways. Hence, fucoidan can be considered as a potential therapeutic reagent against the metastasis of invasive human lung cancer cells.

Cytotoxicity and hemolytic activity of jellyfish Nemopilema nomurai (Scyphozoa: Rhizostomeae) venom

The recent bloom of a giant jellyfish Nemopilema nomurai has caused a danger to sea bathers and fishery damages in the waters of China, Korea, and Japan. The present study investigated the cytotoxic and hemolytic activities of crude venom extract of N. nomurai using a number of in vitro assays. The jellyfish venom showed a much higher cytotoxic activity in H9C2 heart myoblast than in C2C12 skeletal myoblast (LC(50)=2 microg/mL vs. 12 microg/mL, respectively), suggesting its possible in vivo selective toxicity on cardiac tissue. This result is consistent with our previous finding that cardiovascular function is a target of the venom. In order to determine the stability of N. nomurai venom, its cytotoxicity was examined under the various temperature and pH conditions. The activity was relatively well retained at low environmental temperature (<or=20 degrees C) and dramatically lost at high temperature (>or=60 degrees C). In pH stability test, the venom has abruptly lost its activity at low pH environment (pH<or=4). Interestingly enough, however, its activity was not significantly affected even at the highest pH environment tested (pH<or=12) in the present study. Additionally, hemolytic activity of the venom was examined using the erythrocytes of cat, dog, human, rabbit and rat. Venom concentration-dependent hemolysis could be observed from 10 microg/mL of protein equivalents or higher with variable potencies in different species, among which dog erythrocyte was the most susceptible to the venom (EC(50)=151 microg/mL). SDS-PAGE analysis of N. nomurai venom showed the molecules of 20-40 kDa and 10-15 kDa appeared to be the major protein components of the venom.

Brown alga Ecklonia cava attenuates type 1 diabetes by activating AMPK and Akt signaling pathways.

The antidiabetic therapeutic effect of Ecklonia cava, a brown alga, was investigated using streptozotocin-induced type 1 diabetes mellitus rats and C2C12 myoblasts. The methanol extract of E. cava (ECM), having a strong radical scavenging activity, significantly reduced plasma glucose level and increased insulin concentration in type 1 diabetes mellitus rats. Moreover, the elevation of plasma ALT in diabetic rats was dramatically restored near to normal range by the treatment of ECM, whereas AST level was not meaningfully altered in any group throughout the experiment. The characteristic indications of diabetes, such as polyphagia and polydipsia, were greatly improved by ECM treatment as well. The mechanism of action of ECM appears to be, at least partially, mediated by the activation of both AMP-activated protein kinase/ACC and PI-3 kinase/Akt signal pathways. Taken together, the present results suggest that E. cava has both in vivo and in vitro antidiabetic effects.

Evaluation of anti-oxidant and anti-cancer properties of Dendropanax morbifera Léveille.

Dendropanax morbifera Léveille, an endemic species in Korea, is best known as a tree that produces a resinous sap. Although D. morbifera is used in folk medicine, its biological activities are poorly understood. In this study, the methanolic extracts of D. morbifera branches, debarked stems, bark, and two different stages of leaves were evaluated for anti-oxidant activity and anti-cancer potential. The debarked stem extract exhibited strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity and reducing power compared with other samples. In addition, the cytotoxic activities of these extracts were investigated in human tumour cell lines. The results suggested that the extracts of debarked stems, green leaves, and yellow leaves were the potent source of anti-cancer compounds, particularly in Huh-7 cells. Furthermore, treatment with the extracts of debarked stems, green leaves, and yellow leaves caused an increase of apoptotic or senescent cells in Huh-7 cells. Twenty-four hour treatment with debarked stems extract resulted in the strong induction of p53 and p16, whereas both leaf extracts inhibited the activation of ERK. The debarked stems and green leaf extracts reduced Akt levels in Huh-7 cells, indicating that D. morbifera extracts caused the activation of p16 and p53 pathways. This, together with the inhibition of Akt or ERK signalling, resulted in suppression of Huh-7 cell proliferation. These results suggest that methanolic leaf and debarked stems extracts are a source of anti-oxidant and anti-cancer compounds, and could be developed as a botanical drug.

Synergistic effect of curcumin and insulin on muscle cell glucose metabolism

Curcumin is a major phenolic compound of Curcuma longa, which has long been used in traditional Indian medicine. Recently, curcumin has been reported to have antihyperglycemic activity in animal models. However, the molecular basis of this action has not been adequately described. In the present study, curcumin was observed to contain a high level of polyphenols and strong antioxidant activity. Then, the antihyperglycemic effect of curcumin was examined for various signaling pathways using C(2)C(12) mouse myoblast cells. From this, curcumin treatment strongly induced glucose uptake and the phosphorylation of AMPK (AMP-activated protein kinase)/ACC (acetyl-CoA carboxylase), but not PI3-kinase (phosphoinositide 3-kinase)/Akt. Interestingly, the co-treatment of insulin and curcumin produced a mutual synergistic activation of both AMPK/ACC and PI3-kinase/Akt pathways. On the other hand, the synergism could not be observed from the co-treatment of insulin and EGCG. It suggests that the two signaling pathways can crosstalk each other in a case-sensitive manner upon the treatment of antioxidant polyphenols. Further, these results were consistent with the findings of GLUT4 translocation to the cell surface. Our findings indicate that curcumin can promote AMPK activation and glucose uptake with increased insulin sensitivity in muscle cells as a potential anti-diabetic therapeutic agent.

Syntheses and evaluation of pyrido[2,3-d]pyrimidine-2,4-diones as PDE 4 inhibitors

The syntheses and in vitro evaluation of a new series of pyrido[2,3-d]pyrimidine-2,4-diones bearing substituents at C-3 and/or C-4 positions on the pyridine ring are described. Some of these compounds, especially 51 and 6f, were found to be potent phosphodiesterase 4 (PDE 4) inhibitors exhibiting improved ratio of PDE 4 inhibitory activity:rolipram binding assay (RBA).

Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling.

Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100‐kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull‐down assay using GST–Src–SH2 fusion proteins. We found MVP as a Src–SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF‐dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration‐dependent manner. MVP overexpression downregulates EGF‐dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src‐mediated signalling cascades.

Enzymatic analysis of Hemiscorpius lepturus scorpion venom using zymography and venom-specific antivenin.

Hemiscorpius lepturus envenomation exhibits various pathological changes in the affected tissues, including skin, blood cells, cardiovascular and central nervous systems. The enzymatic activity and protein component of the venom have not been described previously. In the present study, the electrophoretic profile of H. lepturus venom was determined by SDS-PAGE (12 and 15%), resulting in major protein bands at 3.5-5, 30-35 and 50-60 kDa. The enzymatic activities of the venom was, for the first time, investigated using various zymography techniques, which showed the gelatinolytic, caseinolytic, and hyaluronidase activities mainly at around 50-60 kDa, 30-40 kDa, and 40-50 kDa, respectively. Among these, the proteolytic activities was almost completely disappeared in the presence of a matrix metalloproteinase inhibitor, 1, 10-phenanthroline. Antigen-antibody interactions between the venom and its Iranian antivenin was observed by Western blotting, and it showed several antigenic proteins in the range of 30-160 kDa. This strong antigen-antibody reaction was also demonstrated through an enzyme-linked immunosorbent assay (ELISA). The gelatinase activity of the venom was suppressed by Razi institute polyvalent antivenin, suggesting the inhibitory effect of the antivenin against H. lepturus venom protease activities. Prudently, more extensive clinical studies are necessary for validation of its use in envenomed patients.

Saffron (Crocus sativus L.) increases glucose uptake and insulin sensitivity in muscle cells via multipathway mechanisms

Saffron (Crocus sativus Linn.) has been an important subject of research in the past two decades because of its various biological properties, including anti-cancer, anti-inflammatory, and anti-atherosclerotic activities. On the other hand, the molecular bases of its actions have been scarcely understood. Here, we elucidated the mechanism of the hypoglycemic actions of saffron through investigating its signaling pathways associated with glucose metabolism in C(2)C(12) skeletal muscle cells. Saffron strongly enhanced glucose uptake and the phosphorylation of AMPK (AMP-activated protein kinase)/ACC (acetyl-CoA carboxylase) and MAPKs (mitogen-activated protein kinases), but not PI 3-kinase (Phosphatidylinositol 3-kinase)/Akt. Interestingly, the co-treatment of saffron and insulin further improved the insulin sensitivity via both insulin-independent (AMPK/ACC and MAPKs) and insulin-dependent (PI 3-kinase/Akt and mTOR) pathways. It also suggested that there is a crosstalk between the two signaling pathways of glucose metabolism in skeletal muscle cells. These results could be confirmed from the findings of GLUT4 translocation. Taken together, AMPK plays a major role in the effects of saffron on glucose uptake and insulin sensitivity in skeletal muscle cells. Our study provides important insights for the possible mechanism of action of saffron and its potential as a therapeutic agent in diabetic patients.

Cytotoxicity and the induction of the stress protein Hsp 70 in Chang liver cells in response to zearalenone-induced oxidative stress

Zearalenone (ZEN) has been implicated in several cases of mycotoxicosis in farm animals and humans. The toxic effects of ZEN have been well characterized, but little is known regarding the mechanisms of ZEN toxicity, including the involvement of the oxidative stress pathway. Using Chang liver cells as a model, the aim of this study was to determine if ZEN could elevate the expression of the heat shock protein Hsp 70, induce cytotoxicity and modulate the levels of glutathione (GSH) and thiobarbituric acid reactive substance (TBARS). In addition, the cytoprotective effects of N-acetylcysteine amide (NACA) pre-treatment were assessed. Finally, the involvement of oxidative stress in ZEN-induced toxicity was confirmed. The results of this study demonstrated that ZEN-induced Hsp 70 expression in a dose- and time-dependent manners. This effect occurred at low-ZEN concentrations, and could therefore be considered a biomarker of ZEN-induced toxicity. The cytotoxicity was reduced when Chang liver cells were exposed to sub-lethal heat shock prior to ZEN treatment, demonstrating a cytoprotective effect of Hsp 70. This cytoprotective effect suggested that Hsp 70 might play a key role in the cellular defense mechanism. When cells were pre-treated with NACA prior to ZEN treatment, the cells were also protected from toxicity. This NACA cytoprotective effect suggested the involvement of oxidative stress in ZEN-induced toxicity, and this mechanism was supported by reduced Hsp 70 expression, inhibited cytolethality, increased GSH levels and decreased TBARS formation when cells were pre-treated with NACA prior to ZEN exposure. Our data clearly demonstrated that ZEN induced cytotoxicity in Chang liver cells by inhibiting cell proliferation, decreasing GSH levels and increasing TBARS formation in a dose-dependent manner. ZEN also, induced Hsp 70 expression, and the side effects of ZEN were significantly alleviated by pre-treatment with NACA. Oxidative stress is likely to be one of the primary pathways of ZEN toxicity. This oxidative stress may contribute, at least in part, to the mechanism of ZEN-induced cytotoxicity.