Novel Midkine Inhibitor Induces Cell Cycle Arrest and Apoptosis in Multiple Myeloma.

BACKGROUND/AIM: Multiple myeloma (MM), the second most common hematological malignancy, is characterized by the accumulation of malignant plasma cells within the bone marrow. Despite various drug classes for MM treatment, it remains incurable, necessitating novel and efficacious agents. This study aims to explore the anti-cancer activity of a midkine inhibitor, iMDK (C21H13FN2O2S), in myeloma cell lines. MATERIALS AND METHODS: This study assessed the antiproliferative activity using the MTT assay. Cell cycle and apoptosis were evaluated using flow cytometry. To further investigate the inhibitory mechanism, western blotting was used to detect cell cycle-related proteins, pro-apoptotic proteins, and anti-apoptotic proteins. RESULTS: iMDK inhibits MM cell proliferation in a dose- and time-dependent manner, inducing cell cycle arrest and apoptosis. The reduction in Cdc20 expression by iMDK treatment leads to G2/M phase cell cycle arrest. Furthermore, iMDK down-regulates anti-apoptotic proteins (Bcl-2, Bcl-xL, Mcl-1, and c-FLIP), thereby activating both intrinsic and extrinsic apoptosis pathways. CONCLUSION: iMDK could be a potential candidate for MM treatment.

Synthesis and structure-activity relationship study of aldose reductase inhibiting marine alkaloid lukianol A and its derivatives.

Lukianol A (1a) and its six derivatives 1b-1g, in which each hydroxyl groups of 1a was individually modified, were synthesized via the common intermediate 7a, which was obtained by condensation of the styryl carbazate 10 with p-hydroxyphenylpyruvic acid and subsequent [3,3]-sigmatropic rearrangement. The synthesized lukianol derivatives were evaluated for their ability to inhibit human aldose reductase. 4'-O-methyl (1b) and 4'-dehydroxy (1g) derivatives showed the same level of inhibitory activity as 1a (IC50 2.2 µm), indicating that the 4'-OH is irrelevant for the activity. In contrast, methylation of the hydroxyl group at the 4″'-position (1d) resulted in the loss of activity at a concentration of 10 µm, and masking the hydroxyl group at the 4″-position (1e) caused a 9-fold decrease in activity compared with that of 1b, suggesting that the 4″-OH is an essential group, and the 4″'-OH is required for higher activity.

Midkine inhibitor (iMDK) induces apoptosis of primary effusion lymphoma via G2/M cell cycle arrest.

Primary effusion lymphoma (PEL) is an aggressive B-cell non-Hodgkin lymphoma in immunocompromised individuals such as AIDS patients. PEL shows a poor prognosis (median survival time < 6 months) compared with other AIDS-related lymphomas, and is generally resistant to conventional treatments. Novel drugs for PEL treatment are required. Midkine inhibitor (iMDK) was previously found to suppress midkine protein expression. Interestingly, iMDK suppressed cell proliferation in PEL cell lines in a time- and dose-dependent manner, regardless of midkine gene expression. We examined the mechanism of iMDK on PEL. Importantly, iMDK strongly induced cell cycle arrest at the G2/M phase within 12 h of incubation and suppressed the p-CDK1 protein level, which is associated with the cell cycle checkpoint at G2/M, resulting in mitotic catastrophe with observation of multipolar division. After mitotic catastrophe, iMDK-treated PEL showed apoptosis with caspase-3, - 8, and - 9 activation at 24 h incubation. However, iMDK showed no effects on viral protein-activated signaling pathways such as JAK-STAT, PI3K-Akt and NF-κB, and HHV-8/KSHV gene expression in PEL. These results indicate that iMDK is a novel CDK1 inhibitor and a promising lead compound for PEL chemotherapy treatment.

Sulfated polysaccharide ascophyllan prevents amyloid fibril formation of human insulin and inhibits amyloid-induced hemolysis and cytotoxicity in PC12 cells.

We found that ascophyllan significantly inhibited the fibrillation of human insulin and was the most effective among the sulfated polysaccharides tested. Gel-filtration analysis suggested that ascophyllan was capable of forming a complex with insulin through a weak interaction. Secondary structure transition from native α-helix to ß-sheet predominant structure of insulin under the fibrillation conditions was suppressed in the presence of ascophyllan. Interestingly, ascophyllan attenuated insulin fibril-induced hemolysis of human erythrocytes. Moreover, ascophyllan attenuated insulin amyloid-induced cytotoxicity on rat pheochromocytoma PC12 cells and reduced the level of intracellular reactive oxygen species. This is the first report indicating that a sulfated polysaccharide, ascophyllan, can suppress the insulin amyloid fibril formation and inhibit the fibril-induced detrimental bioactivities.

Induction of Apoptotic Cell Death in Human Leukemia U937 Cells by C18 Hydroxy Unsaturated Fatty Acid Isolated from Red Alga Tricleocarpa jejuensis.

Our previous studies have found that (±)-(E)-12-hydroxyoctadec-10-enoic acid (HOEA) isolated from the red alga Tricleocarpa jejuensis showed cytotoxic effects on various living organisms including harmful microalgae, Gram-positive bacteria, and mammalian tumor cells. Since natural products with apoptosis-inducing ability can be promising anti-cancer agents, in this study, we investigated the cytotoxic mechanism of HOEA on U937 cells focusing on apoptosis induction. HOEA showed much stronger cytotoxic and cytolytic effects on U937 cells than elaidic acid, which has similar structure but no 12-hydroxy group, suggesting that hydroxy group is important for the cytotoxicity of HOEA. HOEA induced apoptotic nuclear morphological changes, DNA fragmentation, and decrease in mitochondrial membrane potential. Furthermore, time-dependent increase in annexin V+/PI+ cell population in HOEA-treated U937 cells was detected. Among the apoptosis-related reagents, caspase-family inhibitor almost completely inhibited HOEA-induced DNA fragmentation. In the analyses using specific caspase-substrates, extremely high cleavage activity toward caspase-3/7/8 substrate was observed in HOEA-treated U937 cells, and weak activities of caspase-1 and -3 were detected. Analyses using specific caspase inhibitors suggested that caspase-3 and caspase-8 might be predominantly responsible for the cleavage activity. Activation of these caspases were also confirmed by western blotting in which significant levels of cleaved forms of caspase 3, caspase 8, and PARP were detected in HOEA-treated U937 cells. Our results suggest that HOEA is capable of inducing apoptosis in U937 cells in which caspase-3 and caspase-8 might play important roles. Since the cytotoxic effect of HOEA is not strictly specific to tumor cells, development of appropriate drug delivery system for selective tumor targeting is necessary for the clinical applications to reduce the possible side effects.

Cucurbitacin B induces apoptosis of primary effusion lymphoma via disruption of cytoskeletal organization.

BACKGROUND: Primary effusion lymphoma (PEL) is an aggressive B cell non-Hodgkin lymphoma that develops especially in AIDS patients and immunocompromised patients infected with human herpes virus-8 (HHV-8)/Kaposi's sarcoma-associated herpesvirus (KSHV). PEL has a poor prognosis in patients despite conventional chemotherapeutic treatment, and a safe and efficient therapy is required. PURPOSE: To examine the effects on PEL of cucurbitacin B (CuB), a triterpene found in plants of the Cucurbitaceae family that has several anti-cancer activities. STUDY DESIGN: We evaluated the anti-cancer activities of CuB in vitro and in vivo. METHODS: Cell proliferation of PEL cell lines was measured by MTT assay. Cleaved caspases and signaling transduction associated proteins were analyzed by western blotting. Wright and Giemsa staining and immunofluorescence staining were carried out to observe cell morphology. Cell cycles were analyzed by flow cytometry. RT-PCR was performed to detect viral gene expressions. A xenograft mouse model was employed to evaluate the anti-cancer activity of CuB in vivo. RESULTS: CuB inhibited cell proliferation of PEL cell lines (BCBL-1, BC-1, GTO and TY-1) in a dose-dependent manner (0-50 nM) and induced apoptosis of BCBL-1 cells via caspase activation in a dose- and time-dependent manner. In addition, CuB caused cell-shape disruption by inducing actin aggregation and suppressing the p-cofilin level, resulting in BCBL-1 cell arrest at the G2/M phase. In contrast, CuB showed almost no suppression of p-STAT3 and p-Akt activation, which were constitutively activated by KSHV-derived proteins. Furthermore, CuB (0.5 mg/kg) via intraperitoneal injection significantly (p < 0.05) suppressed solid tumor growth in the xenograft mouse model. CONCLUSION: This study suggests that CuB is a promising agent for PEL treatment.

Acidic polysaccharides isolated from marine algae inhibit the early step of viral infection.

We examined the effects of various acidic polysaccharides isolated from marine algae on the infection and replication of human immunodeficiency virus type-1 (HIV-1), hepatitis B virus (HBV), hepatitis C virus (HCV), and human T-cell leukemia virus type-1 (HTLV-1). It was found that sulfated fucan polysaccharides, ascophyllan, and two fucoidans derived from different sources significantly inhibited the early step of HIV-1 (R9 and JR-FL) infection, while they did not affect the late step. The alginate oligomer consisted of uronic acids and sulfated-galactan porphyran showed no significant inhibitory effects. In addition, ascophyllan and two fucoidans inhibited the early step of HBV infection in a dose-dependent manner. Furthermore, these polysaccharides inhibited the early step of HCV infection but had no inhibitory effects on HTLV-1 replication. To further examine the specificity of these polysaccharides in viral infections, we used vesicular stomatitis virus (VSV)-G-pseudotyped HIV-1 infection. Ascophyllan, the two fucoidans, and alginate oligomer also potently inhibited VSV-G-pseudotyped HIV-1 infection in HeLa cells. Taken together, these results suggest that the acidic polysaccharides used in this study are capable of inhibiting the early step of viral infections depending on the polysaccharides but not in a strict species-specific manner.

Apolipoprotein E is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages.

Apolipoprotein E (ApoE) belongs to a class of cellular proteins involved in lipid metabolism. ApoE is a polymorphic protein produced primarily in macrophages and astrocytes. Different isoforms of ApoE have been associated with susceptibility to various diseases including Alzheimer's and cardiovascular diseases. ApoE expression has also been found to affect susceptibility to several viral diseases, including Hepatitis C and E, but its effect on the life cycle of HIV-1 remains obscure. In this study, we initially found that HIV-1 infection selectively up-regulated ApoE in human monocyte-derived macrophages (MDMs). Interestingly, ApoE knockdown in MDMs enhanced the production and infectivity of HIV-1, and was associated with increased localization of viral envelope (Env) proteins to the cell surface. Consistent with this, ApoE over-expression in 293T cells suppressed Env expression and viral infectivity, which was also observed with HIV-2 Env, but not with VSV-G Env. Mechanistic studies revealed that the C-terminal region of ApoE was required for its inhibitory effect on HIV-1 Env expression. Moreover, we found that ApoE and Env co-localized in the cells, and ApoE associated with gp160, the precursor form of Env, and that the suppression of Env expression by ApoE was cancelled by the treatment with lysosomal inhibitors. Overall, our study revealed that ApoE is an HIV-1-inducible inhibitor of viral production and infectivity in macrophages that exerts its anti-HIV-1 activity through association with gp160 Env via the C-terminal region, which results in subsequent degradation of gp160 Env in the lysosomes.

Increase in anti-inflammatory activities of radical-degraded porphyrans isolated from discolored nori (Pyropia yezoensis).

The anti-inflammatory properties of porphyrans (D1-D4) obtained from four discolored nori (Pyropia yezoensis) with different growth backgrounds were studied to examine possible variations in their bioactivities. Elution profiles of the porphyrans on Sepharose 4B indicated that D2-porphyran had relatively lower-molecular-size porphyrans than the other porphyrans. Inhibitory activities of the four porphyrans against nitric oxide (NO) and tumor necrosis factor-α (TNF-α) secretion by lipopolysaccharide (LPS)-stimulated RAW264.7 cells were different, whereas no significant differences were observed in the sulfate and anhydrogalactose levels. D2-porphyran showed the highest inhibitory activity against NO and TNF-α secretion by LPS-stimulated RAW264.7 cells, whereas D3- and D4-porphyrans had almost no activity. All porphyrans were efficiently degraded by free radical generated with ascorbate and hydrogen peroxide. The free-radical degradation resulted in a significant increase in the inhibitory activities of the four porphyrans against NO and TNF-α secretion, with varying rates depending on the porphyrans. The ability of D2-porphyran to suppress the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells was also significantly enhanced after degradation. Our results suggest that molecular size is an important factor affecting the anti-inflammatory activity of porphyrans, and radical degradation might be a promising procedure to obtain active low-molecular-size porphyrans.

Inhibitory effect of sulphated polysaccharide porphyran (isolated from Porphyra yezoensis) on RANKL-induced differentiation of RAW264.7 cells into osteoclasts.

Safe and efficient therapeutic agents for bone diseases are required in natural sources. We previously found that edible seaweed-derived polysaccharide porphyran exhibited anti-inflammatory effects through the down regulation of nuclear factor-κB. The aim of this study was to investigate the availability of porphyran as a therapeutic agent for bone diseases. The effects of porphyran on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells were examined. Porphyran suppressed RANKL-induced osteoclast formation in a concentration-dependent manner (6.25-50 µg/ml) without any cytotoxic effects. Furthermore, real-time polymerase chain reaction analyses indicated that porphyran at 50 µg/ml significantly attenuated the RANKL-induced increase in the mRNA levels of osteoclastogenesis-related marker genes such as nuclear factor of activated T cells, tartrate-resistant acid phosphatase, cathepsin K, and matrix metalloproteinase-9 in RAW264.7 cells. To our knowledge, this is the first report showing that edible-seaweed-derived polysaccharide porphyran can suppress RANKL-induced osteoclastogenesis. Our results suggest that porphyran can be used as a safe therapeutic agent to improve osteoclast-related pathological conditions.

Macrophage-stimulating activities of newly isolated complex polysaccharides from Parachlorella kessleri strain KNK-A001.

Our previous studies demonstrated that the microalga Parachlorella kessleri (KNK-A001) has immunostimulatory activities, which were observed as an increase in natural killer (NK) cell activity in mice after intraperitoneal injection or as a protective effect on a virus-infected model shrimp after oral administration. In this study, we attempted to gain insight into the constituent substances of KNK-A001 that are responsible for the immunostimulatory activity. First, we obtained five polysaccharide fractions from KNK-A001 by DEAE anion exchange chromatography. Among the fractions, F5 showed the most potent induction of nitric oxide (NO) secretion in RAW264.7 cells, and both mRNA and protein expression levels of inducible NO synthase (iNOS) were increased in F5-treated RAW264.7 cells. A significant increase in the nuclear translocation of the p65 subunit of nuclear factor-kappa B (NF-κB) was observed in F5-treated RAW264.7 cells. F5 also induced the secretion of tumor necrosis factor (TNF)-α in RAW264.7 cells. Analysis using mitogen-activated protein (MAP) kinase inhibitors suggested that c-Jun N-terminal kinase (JNK) and p38 MAP kinase were mainly involved in F5-induced NO and TNF-α productions. The compositional analysis of F5 identified the main constituents as galactose, glucose, galacturonic acid, and mannose. Gel-filtration analysis suggested that molecular mass of F5 was approximately 400kDa.

Effects of alginate oligomer on the expression of cell cycle- and stress-related genes in Chlamydomonas reinhardtii.

Enzymatically prepared alginate oligomer (AO) promoted the growth of Chlamydomonas reinhardtii in a concentration-dependent manner. AO at 2.5 mg/mL induced increase in expression levels of cyclin A, cyclin B, and cyclin D in C. reinhardtii. CuSO4 at 100 µM suppressed the growth of C. reinhardtiin, and AO at 2.5 mg/mL significantly alleviated the toxicity of CuSO4. Increased intracellular reactive oxygen species level in C. reinhardtii induced by CuSO4 was reduced by AO. After cultivation with CuSO4 at 100 µM, expression levels of ascorbate peroxidase and superoxide dismutase in C. reinhardtii were increased, and AO reduced the increased levels of these enzymes. These results suggest that AO exhibits beneficial effects on C. reinhardtii through influencing the expression of various genes not only at normal growth condition but also under CuSO4 stress.

Protective effect of porphyran isolated from discolored nori (Porphyra yezoensis) on lipopolysaccharide-induced endotoxin shock in mice.

Porphyran, a sulfated polysaccharide, isolated from discolored nori (Porphyra yezoensis) (dc-porphyran) and one fraction (F1) purified from dc-porphyran by DEAE-chromatography showed the protective effects on LPS-induced endotoxin shock in mice. Intraperitoneal (i.p.) treatment with dc-porphyran or F1 (100mg/kg) 60min prior to i.p. injection of LPS (30mg/kg) completely protected mice from LPS lethality. At 10mg/kg concentration, F1 demonstrated more protection than dc-porphyran. Intravenous (i.v.) challenge of LPS, even at 20mg/kg, was more lethal than i.p. administration; i.v. injection of F1 (100mg/kg) with LPS significantly improved the survival rate. However, i.v. dc-porphyran (100mg/kg) produced an even lower survival rate than that of LPS alone. We examined pro-inflammatory mediators such as NO and TNF-α in serum. F1 significantly reduced the levels of these markers. Additionally, F1 significantly decreased the malondialdehyde level in the liver, a marker of oxidative stress, while dc-porphyran had almost no effect. Furthermore, F1 significantly decreased the production of TNF-α and NO in peritoneal exudate cells harvested from LPS-challenged mice, while dc-porphyran treatment showed a lesser decrease. Our results suggest that porphyran isolated from discolored nori, especially F1, is capable of suppressing LPS-induced endotoxin shock in vivo.

Alginate oligomer induces nitric oxide (NO) production in RAW264.7 cells: elucidation of the underlying intracellular signaling mechanism.

Alginate is an acidic linear polysaccharide with immune-modulating activities. In this study, we found that enzymatically digested alginate oligomer (AO) with various degrees of polymerization (DP; 2-5) induced a higher level of nitric oxide (NO) production in RAW264.7 cells than undigested alginate polymer (AP). Reverse transcription-polymerase chain reaction and western blot analyses revealed that the expression level of inducible NO synthase in AO-treated RAW264.7 cells was higher than that in AP-treated cells. AO induced nuclear translocation of nuclear factor (NF)-κB p65 subunit in RAW264.7 cells to a greater extent than AP. Although AO and AP induced similar extents of phosphorylation in three mitogen-activated protein (MAP) kinases, c-Jun N-terminal kinase inhibitor exhibited the most potent inhibitory effect on NO induction in AO- and AP-treated RAW264.7 cells, among three MAP kinase inhibitors that were tested.

Anti-metastatic effects of the sulfated polysaccharide ascophyllan isolated from Ascophyllum nodosum on B16 melanoma.

We previously found that ascophyllan, a sulfated polysaccharide isolated from brown seaweed Ascophyllum nodosum, exhibited antitumor activity in sarcoma-180 tumor-bearing mice. In this study, we found that ascophyllan inhibited the migration and adhesion of B16 melanoma cells by reducing the expression of N-cadherin and enhancing the expression of E-cadherin in a concentration-dependent manner. Transwell invasion assay revealed that ascophyllan suppressed the invasion ability of B16 cells. It also inhibited the expression of matrix metalloprotease-9 (MMP-9) mRNA and the secretion of MMP-9 protein in B16 cells, a process that may involve the extracellular signal-regulated kinase (ERK) signaling pathway. Furthermore, ascophyllan administered intraperitoneally at 25 mg/kg showed anti-metastatic activity in a mouse model of metastasis induced by intravenous injection of B16 cells, and the number of lung surface metastatic nodules in ascophyllan-treated mice was significantly reduced compared to that in the untreated control mice. Since splenic natural killer cell activity enhanced in the mice injected with ascophyllan intraperitoneally, we suggest that ascophyllan may exhibit in vivo anti-metastatic activity on B16 melanoma cells through activation of the host immune system in addition to a direct action on cancer cells.

Antioxidant and anti-inflammatory activities of porphyran isolated from discolored nori (Porphyra yezoensis).

We found that discolored waste nori with no commercial value, contains much higher level of porphyran than normal nori that is a sheeted food stuff prepared from P. yezoensis used in sushi. Chemical analyses revealed that mean molecular mass of the porphyran prepared from discolored nori (dc-porphyran) was much lower than that of the porphyran from normal nori (n-porphyran). Dc-porphyran showed slightly greater scavenging activity toward superoxide anion and hydroxyl radical than n-porphyran. Dc-porphyran inhibited nitric oxide (NO) production in LPS-stimulated RAW264.7 cells through preventing the expression of inducible NO synthase, whereas no such activity was observed in n-porphyran. Since acid-hydrolyzed n-porphyran showed the inhibitory activity on NO production from LPS-stimulated RAW264.7 cells, the molecular size of porphyran was suggested to be a critical factor for the activity. Dc-porphyran was separated into 4 fractions (F1-F4) on DEAE-chromatography, and F1 showed the highest inhibitory effect on NO production from LPS-stimulated RAW264.7 cells. Our results indicate that discolored waste nori is useful as a source of porphyran with even better bioactivities than porphyran from normal nori.

Biological activities of alginate.

To gain insight into the structure-activity relationship of alginate, we examined the effect of alginates with varying molecular weights and M/G ratio on murine macrophage cell line, RAW264.7 cells in terms of induction of tumor necrosis factor-α (TNF-α) secretion. Among the alginates tested, alginate with the highest molecular weight (MW 38,000, M/G 2.24) showed the most potent TNF-α-inducing activity. Alginates having higher M/G ratio tended to show higher activity. These results suggest that molecular size and M/G ratio are important structural parameters influencing the TNF-α-inducing activity. Interestingly, enzymatic depolymerization of alginate with bacterial alginate lyase resulted in dramatic increase in the TNF-α-inducing activity. The higher activity of enzymatically digested alginate oligomers to induce nitric oxide production from RAW264.7 cells than alginate polymer was also observed. On the other hand, alginate polymer and oligomer showed nearly equal hydroxyl radical scavenging activities.

Inhibitory effect of orally-administered sulfated polysaccharide ascophyllan isolated from ascophyllum nodosum on the growth of sarcoma-180 solid tumor in mice.

We evaluated the antitumor activity of crude extract and ascophyllan prepared from Ascophyllum nodosum in sarcoma-180 solid tumor-bearing mice with continuous intraperitoneal (i.p.) administration at a dose of 50 mg/kg body weight/day or oral administration at a dose of 500 mg/kg body weight/day. Ascophyllan and crude extract administered via the oral route showed greater antitumor effects than via i.p. route, and the tumor sizes in mice treated with ascopyllan and crude extract were reduced by a mean of 68.7±6.8% and 42.4±24.8% by the oral route, and 41.4±16.1% and 13.6±20.6% by i.p. route compared to control mice. Splenic natural killer cell activity in the mice treated with ascophyllan and crude extract by i.p. route was significantly enhanced, while only a slight increase of this activity was observed in orally-treated mice. Furthermore, increase in spleen weight of tumor-bearing mice was slightly suppressed by oral administration of ascophyllan, whereas i.p. administration resulted in further enlargement. Analysis of serum cytokines revealed that oral treatment with ascophyllan resulted in significant increase of tumor necrosis factor-α and interleukin-12 levels. Since ascophyllan showed no direct cytotoxic effect on sarcoma-180 cells, orally-administered ascophyllan is suggested to exhibit its antitumor activity through the activation of the host immune system.

Importance of sulfate groups for the macrophage-stimulating activities of ascophyllan isolated from the brown alga Ascophyllum nodosum.

To investigate the role of sulfate groups on the macrophage-stimulating activities of ascophyllan, we prepared desulfated ascophyllan, and its effects on RAW264.7 cells were compared with native ascophyllan. The chemical structural analysis revealed that nearly 21% of sulfate groups of ascophyllan were removed by desulfation reaction, while no significant changes in the molecular mass and monosaccharide composition occurred after desulfation. NO- and cytokine- (TNF-α and G-CSF) inducing activities of the desulfated ascophyllan on RAW264.7 cells were significantly decreased as compared to native ascophyllan. Furthermore, the activity of desulfated ascophyllan to induce reactive oxygen species (ROS) generation from RAW264.7 cells decreased to almost negligible level. Our results suggest that the level of sulfate groups of ascophyllan is an important structural element responsible for the macrophage-stimulating activities. Probably, even the limited removal of sulfate residues sensitive to desulfation reaction may result in significant decrease in the bioactivities of ascophyllan.

Stimulatory effect of the sulfated polysaccharide ascophyllan on the respiratory burst in RAW264.7 macrophages.

In this study, we investigated the bioactivity of ascophyllan in terms of reactive oxygen species (ROS) generation. In RAW264.7 cells, we found that ascophyllan induced ROS generation in a concentration-dependent manner, but with bell-shaped profile. Immunoblot analysis demonstrated that ascophyllan promoted the translocation of cytosolic subunits (p67(phox) and p47(phox)) of NADPH oxidase to the plasma membrane. Among mitogen activated protein (MAP) kinase inhibitors tested, JNK inhibitor showed the most potent inhibitory effect on ascophyllan-induced ROS production. Consistently, significant level of phosphorylated JNK MAP kinase was detected in ascophyllan-treated RAW264.7 cells. Our findings suggest for the first time that ascophyllan can stimulate macrophages to produce ROS through the activations of NADPH oxidase and JNK MAP kinase.