No abstract

Fucoidan is a sulfated polysaccharide found in edible brown algae, such as Undaria pinnatifida, Fucus vesiculosus and Ecklonia cava. Fucoidan usually contains a large proportion of L-fucose and sulfate. Fucoidan has been reported to show various biological activities such as anti-tumor, [1][2][3][4] anti-coagulant, 5,6) anti-viral, 7) and anti-inflammatory. 8) Furthermore, its anti-tumor activity may be due to the inhibition of tumor angiogenesis in Ehrlich ascites carcinoma 1) and lung carcinoma, 9) as well as the direct induction of apoptosis in U9373) and HS-sultan cells. 4) Several marine algal polysaccharides, fucoidan in particular, have been found to induce apoptosis in cancer cells. [10][11][12] Nevertheless, there is no report on the effect of fucoidan in colon cancer, one of the most malignant neoplasias and a frequently occurring tumor in the world.Recently, it has been demonstrated that the phosphorylation/de-phosphorylation states of some regulatory proteins are crucial events along the pathways controlling cell growth and apoptosis. A well-established apoptotic signaling cascade is regulated by mitogen activated protein (MAP) kinases.13) The MAPK pathway consists of a three-tiered kinase core where MAP3K activates an MAP2K which in turn activates an MAPK (ERK, JNK; c-Jun N-terminal kinase, and p38), resulting in the activation of nuclear factor-kB (NF-kB) and cell survival.14,15) Akt signaling is another important transduction pathway that plays a critical role in controlling the balance between cell survival and apoptosis. 16) In this study, we investigated the effect of fucoidan on the induction of apoptosis in HCT-15 cells, human colon adenocarcinoma cells. Because MAPK and PI3K/Akt pathways are involved in cellular proliferation, differentiation, and apoptosis, [17][18][19][20] the phosphorylation and activities of two MAPKs, ERK and p38 MAPK as well as Akt were investigated. Understanding of the underlying mechanism of the induction of apoptosis by fucoidan will benefit the development of chemopreventive and/or chemotherapeutics for colon cancer. MATERIALS AND METHODS Fucoidan and Diallyl DisulfideFucoidan (from Fucus vesiculosus) and diallyl disulfide (DADS) were purchased from Sigma (St. Louis, MO, U.S.A.). The fucoidan was dissolved in phosphate-buffered saline (PBS; Sigma, St. Louis, MO, U.S.A.) to 50 mg/ml and the DADS was dissolved in dimethylsulfoxide (DMSO; Sigma, St. Louis, MO, U.S.A.) to 50 mM at Ϫ20°C until further use.Cell Culture The HCT-15 human colon cancer cells were purchased from the Korea Cell Line Bank (KCLB) and cultured in RPMI1640 (Gibco BRL, Grand Island, NY, U.S.A.) medium supplemented with 10% fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, U.S.A.) at 37°C in a 5% CO 2 atmosphere. The exponentially growing cells were used throughout the experiments.MTT Assay The 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma, Saint Louis, MO, U.S.A.) assay was performed as previously described. 21) In brief, HCT-15 cells were cultured in a 96-well pla...

In the present study, a toxic mechanism of silver nanoparticles (AgNPs) was investigated in the nematode, Caenorhabditis elegans, focusing on the involvement of oxidative stress in reproduction toxicity. Initially, AgNPs were tested as potential oxidative stress inducers, and increased formation of reactive oxygen species (ROS) was observed in AgNP-exposed C. elegans. Subsequently, the potential upstream signaling pathway activated in response to AgNP exposure was investigated, paying special attention to the C. elegans PMK-1 p38 mitogen-activated protein kinase (MAPK). Increased PMK-1 p38 MAPK gene and protein expressions were observed in C. elegans exposed to AgNPs. Expression of the p38-dependent transcription factor genes and glutathione S-transferase (GST) enzyme activity was also investigated in wildtype (N2) and pmk-1 mutant (km25) C. elegans exposed to AgNPs. The results indicated that AgNP exposure led to increased ROS formation, increased expression of PMK-1 p38 MAPK and hypoxia-inducible factor (HIF-1), GST enzyme activity, and decreased reproductive potential in wildtype (N2) C. elegans; whereas in the AgNP-exposed pmk-1 (km25) mutant, ROS formation and HIF-1 and GST activation were not observed, and decreased reproductive potential was rescued. These results suggest that oxidative stress is an important mechanism of AgNP-induced reproduction toxicity in C. elegans, and that PMK-1 p38 MAPK plays an important role in it. The results also suggest that GST and HIF-1 activation by AgNP exposure are PMK-1 p38 MAPK-dependent, and that both play an important role in the PMK-1 p38 MAPK-mediated defense pathway to AgNP exposure in C. elegans.

Fucoidan, a sulfated polysaccharide, has a variety of biological activities, such as anti-cancer, anti-angiogenic and anti-inflammatory. However, the mechanisms of action of fucoidan as an anti-cancer agent have not been fully elucidated. The present study examined the anti-cancer effect of fucoidan obtained from Undaria pinnatifida in PC-3 cells, human prostate cancer cells. Fucoidan induced the apoptosis of PC-3 cells by activating both intrinsic and extrinsic pathways. The induction of apoptosis was accompanied by the activation of extracellular signal-regulated kinase mitogen-activated protein kinase (ERK1/2 MAPK) and the inactivation of p38 MAPK and phosphatidylinositol 3-kinase (PI3K)/Akt. In addition, fucoidan also induced the up-regulation of p21Cip1/Waf and down-regulation of E2F-1 cell-cycle-related proteins. Furthermore, in the Wnt/β-catenin pathway, fucoidan activated GSK-3β that resulted in the decrease of β-catenin level, followed by the decrease of c-myc and cyclin D1 expressions, target genes of β-catenin in PC-3 cells. These results suggested that fucoidan treatment could induce intrinsic and extrinsic apoptosis pathways via the activation of ERK1/2 MAPK, the inactivation of p38 MAPK and PI3K/Akt signaling pathway, and the down-regulation of Wnt/β-catenin signaling pathway in PC-3 prostate cancer cells. These data support that fucoidan might have potential for the treatment of prostate cancer.

The p53-inducible gene 3 (PIG3) is originally isolated as a p53 downstream target gene, but its function remains unknown. Here, we report a role of PIG3 in the activation of DNA damage checkpoints, after UV irradiation or radiomimetic drug neocarzinostatin (NCS). We show that depletion of endogenous PIG3 sensitizes cells to DNA damage agents, and impaired DNA repair. PIG3 depletion also allows for UV-and NCS-resistant DNA synthesis and permits cells to progress into mitosis, indicating that PIG3 knockdown can suppress intra-S phase and G2/M checkpoints. PIG3-depleted cells show reduced Chk1 and Chk2 phosphorylation after DNA damage, which may directly contribute to checkpoint bypass. PIG3 exhibited diffuse nuclear staining in the majority of untreated cells and forms discrete nuclear foci in response to DNA damage. PIG3 colocalizes with c-H2AX and 53BP1 to sites of DNA damage after DNA damage, and binds to a c-H2AX. Notably, PIG3 depletion decreases the efficient induction and maintenance of H2AX phosphorylation after DNA damage. Moreover, PIG3 contributes to the recruitment of 53BP1, Mre11, Rad50 and Nbs1 to the sites of DNA break lesions in response to DNA damage. Our combined results suggest that PIG3 is a critical component of the DNA damage response pathway and has a direct role in the transmission of the DNA damage signal from damaged DNA to the intra-S and G2/M checkpoint machinery in human cells.

Although Korea is one of the endemic areas for hepatitis B virus infection (HBV), the prevalence of deletions in HBV preS region occurring naturally have not been determined. In the present study, the prevalence of preS deletions was determined in terms of clinical state and HBeAg serostatus in 120 patients with different clinical features [59 HBeAg positive, 61 HBeAg negative; 38 asymptomatic carriers, 21 patients with chronic hepatitis, 21 patients with liver cirrhosis, 40 patients with hepatocellular carcinoma (HCC)]. A total of 37 strains (30.8%) harbored deletions in the preS region. Overall, the frequencies of preS deletions tended to increase gradually according to the degree of the clinical severity of liver disease. The prevalence of preS1 deletions in HCC patients tended to be higher than in patients with liver cirrhosis (32.5% vs. 19%). The prevalence of preS2 deletions in HBeAg negative patients was significantly higher than in HBeAg positive patients (23% vs. 6.8%). The type of deletion encountered most frequently was one disrupting the preS1 start codon [14/37 strains (37.8%)], which showed a very high prevalence in HCC patients (9/40, 22.5%; HCC vs. asymptomatic carriers, P=0.048). These results suggest that there might be the discrepancy between preS1 and preS2 mutations in the mechanism of enhancing the progression of chronic liver disease, especially the development of HCC and to maintain tolerance during the stage of immune tolerance. Specific deletion of the type disrupting preS1 start codon may play important roles in hepatocarcinogenesis, at least in Korean patients with chronic HBV infection.

Naturally occurring triterpenoid compounds have long been used as anti-inflammatory, antimalarial, and insecticidal agents. It has become evident that some of the natural or synthetic triterpenoids have promising clinical potential as both a therapeutic and chemopreventive agent for cancer. However, the molecular basis for the antitumor activity of triterpenoid has yet to be defined. In this study, we show that pristimerin, a natural triterpenoid, induces mitochondrial cell death in human cervical cancer cells and that reactive oxygen species (ROS)-dependent activation of both Bax and poly(ADP-ribose) polymerase-1 (PARP-1) is critically required for the mitochondrial dysfunction. We also showed that c-Jun N-terminal kinase (JNK) is involved in ROS-dependent Bax activation. Treatment of pristimerin induced an increase in intracellular ROS, JNK activation, conformational change, and mitochondrial redistribution of Bax, mitochondrial membrane potential loss, and cell death. The PARP-1 was also found to be activated by pristimerin treatment. An antioxidant, N-acetyl-L-cysteine (NAC), inhibited pristimerin-induced JNK activation, Bax relocalization, and PARP-1 activation, as well as mitochondrial cell death. Moreover, inhibition of JNK clearly suppressed conformational change and mitochondrial translocation of Bax and subsequent mitochondrial cell death but did not affect PARP-1 activation. Inhibition of PARP-1 with 1,5-dihydroxyisoquinoline (DIQ) or with small interfering RNA of PARP-1 significantly attenuated pristimerininduced mitochondrial membrane potential loss and cell death but did not affect JNK activation and Bax relocalization. These results indicate that the natural triterpenoid pristimerin induces mitochondrial cell death through ROS-dependent activation of both Bax and PARP-1 in human cervical cancer cells and that JNK is involved in ROS-dependent Bax activation.

Aim: To investigate the anti-oxidant properties of esculetin (6,7-dihydroxycoumarin) against H 2 O 2 -induced Chinese hamster lung fibroblast (V79-4) damage. Methods: The radical scavenging activity was assessed by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydroxyl radical, and intracellular reactive oxygen species (ROS). In addition, lipid peroxidation was assayed by the measure of related substances which react with thiobarbituric acid. The amount of carbonyl formation in protein was determined using a protein carbonyl ELISA kit. As well, cellular DNA damage was detected by Western blot and immunofluorescence image. Cell viability was assessed by 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: Esculetin exhibited DPPH radical scavenging, hydroxyl radical scavenging, and intracellular ROS scavenging activities. The radical scavenging activity of esculetin resulted in the protection of cells from lipid peroxidation, protein carbonyl, and DNA damage induced by H 2 O 2 . Therefore, esculetin recovered cell viability exposed to H 2 O 2 . Conclusion: Esculetin efficiently attenuated the oxidative stress induced cell damage via its anti-oxidant properties. As a result, esculetin may be useful in the development of functional food and raw materials of medicine.