• Advances in Traditional Medicine
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• v.7 no.4
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• pp.341-347
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• 2007

Red seaweed (Callophyllis japonica) has long formed part of the diet of Asians, but the pharmacological properties of this plant have not been evaluated. In this study, we examined the effect of an ethanol extract of C. japonica on the generation of nitric oxide (NO) in RAW 264.7 cells. The C. japonica extract increased the generation of NO and tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$), which were detected by the Griess method and an enzyme-linked immunosorbent assay, respectively. The increased production of NO by C. japonica extract was inhibited by $N^G$-monomethyl-L-arginine ($100{\mu}M$), a specific inhibitor of NO production in the L-arginine-dependent pathway, and by the nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) inhibitor, pyrrolidine dithiocarbamate ($10-100{\mu}M$) in a dose-dependent manner. These findings demonstrate that C. japonica extract stimulates the production of NO and $TNF-{\alpha}$ in RAW 264.7 cells through the activation of $NF-{\kappa}B$ and that this extract might also inhibit the growth of the human leukemic cells.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.6
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• pp.2251-2256
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• 2015

To study effects of cellular FLICE (FADD-like IL-$1{\beta}$-converting enzyme)-inhibitory protein (c-FLIP) inhibition by RNA interference (RNAi) on sensitivity of U2OS cells to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-induced apoptosis, plasmid pSUPER-c-FLIP-siRNA was constructed and then transfected into U2OS cells. A stable transfection cell clone U2OS/pSUPER-c-FLIP-siRNA was screened from the c-FLIP-siRNA transfected cells. RT-PCR and Western blotting were applied to measure the expression of c-FLIP at the levels of mRNA and protein. The results indicated that the expression of c-FLIP was significantly suppressed by the c-FLIP-siRNA in the cloned U2OS/pSUPER-c-FLIP-siRNA as compared with the control cells of U2OS/pSUPER. The cloned cell line of U2OS/pSUPER-c-FLIP-siRNA was further examined for TRAILinduced cell death and apoptosis in the presence of a pan-antagonist of inhibitor of apoptosis proteins (IAPs) AT406, with or without 4 hrs pretreatment with rocaglamide, an inhibitor of c-FLIP biosynthesis, for 24 hrs. Cell death effects and apoptosis were measured by the methods of MTT assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and flow cytometry, respectively. The results indicated that TRAIL-induced cell death in U2OS/pSUPER-c-FLIP-siRNA was increased compared with control cells U2OS/pSUPER in the presence or absence of AT406. Flow cytometry indicated that TRAIL-induced cell death effects proceeded through cell apoptosis pathway. However, in the presence of rocaglamide, cell death or apoptotic effects of TRAIL were similar and profound in both cell lines, suggesting that the mechanism of action for both c-FLIP-siRNA and rocaglamide was identical. We conclude that the inhibition of c-FLIP by either c-FLIP-siRNA or rocaglamide can enhance the sensitivity of U2OS to TRAIL-induced apopotosis, suggesting that inhibition of c-FLIP is a good target for anti-cancer therapy.

• Journal of Oriental Neuropsychiatry
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• v.19 no.3
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• pp.117-127
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• 2008

Objective: Bee venom (BV) has been used for the treatment of inflammatory diseases such as rheumatoid arthritis and relief of pain in Oriental medicine. The two main components of BV are melittin and phospholipase A2 (PLA2). Of these, melittin, the major active ingredient of BV, has been reported to induce apoptosis and to possess anti tumor effects. Several studies have established that the agents inducing apoptosis in target organs suppress tumorigenesis. As the other component, PLA2 has been reported to induce neurite outgrowth in PC12 cells. However, there was no report about proliferative effect of BV in neuronal cells. In order to examine the effect of BV on glioma cell, human glioma cell line, U87 was used. Methods: Analysis of proliferation was confirmed by MTT assay. BV increased cell number through dose and duration dependent manner and these effects are apparent at a concentration of 10 ug/ml. To observe which signaling molecules will be activated by BV, phosphorylation of Akt, MAPK, PYK2 or CREB were examined by Western blot analysis. To study the long term effect of BV in U87 cells, the image of cells treated with BV for 4 days were obtained. Results: The phosphorylation levels of PYK2 and Akt were increased at 5 min after addition of 10 ug/ml of BV and sustained to 2 hours. On the other hand, phosphorylation of MAPK and CREB were increased at 5 min, maximum at 10 min, and returned to 30 min. These imply that BV may activate two different signaling pathways, PYK2/Akt and MAPK/CREB. BV treated cells showed increased neurite number and length. Conclusion: These results propose that BV may induce differentiation as well as proliferation of U87 cells through the activation of PYK2/ Akt and MAPK/ CREB.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.21
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• pp.9211-9216
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• 2014

CXCR7 is involved in tumor development and metastasis in multiple malignancies. However, the function and molecular mechanisms of action of CXCR7 in human cervical cancer are still unclear. In the present study a loss of-function approach was used to observe the effects of recombinant CXCR7 specific small interfering RNA pBSilence1.1 plasmids on biological behavior including proliferative activity and invasive potential, as indicated by MTT assays with the cervical cancer SiHa cell line in vitro. Reverse transcription polymerase chain reaction and Western blotting revealed that CXCR7 was downregulated in transfected compared with control cells, associated with inhibited cell growth, invasiveness and migration. The expression of CXCR7 and CXCL12 was also determined immunohistochemically in 152 paraffin-embedded, cervical squamous cell carcinoma (CSCC) and cervical intraepithelial neoplasia (CIN), or normal cervical epithelial to assess clinico-pathological pattern and CXCR7 status with respect to cell differentiation and lymph node metastasis in Uighur patients with CSCC. CXCR7 and CXCL12 expression was higher in cervical cancer than CIN and normal cervical mucosa, especially in those with higher stage and lymph node metastasis. CXCL12 appeared to be positively regulated by CXCR7 at the post-transcriptional level in CSCC. We propose that aberrant expression of CXCR7 plays a role in carcinogenesis, differentiation and metastasis of CSCC, implying its use as a potential target for clinical biomarkers in differentiation and lymph node metastasis.

• Journal of Pharmacopuncture
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• v.20 no.1
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• pp.52-56
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• 2017

Objectives: Neutrophils represent the front line of human defense against infections. Immediately after stimulation, neutrophilic enzymes are activated and produce toxic mediators such as pro-inflammatory cytokines, nitric oxide (NO) and myeloperoxidase (MPO). These mediators can be toxic not only to infectious agents but also to host tissues. Because flavonoids exhibit antioxidant and anti-inflammatory effects, they are subjects of interest for pharmacological modulation of inflammation. In the present study, the effects of rutin on stimulus-induced NO and tumor necrosis factor $(TNF)-{\alpha}$ productions and MPO activity in human neutrophils were investigated. Methods: Human peripheral blood neutrophils were isolated using Ficoll-Hypaque density gradient centrifugation coupled with dextran T500 sedimentation. The cell preparations containing > 98% granulocytes were determined by morphological examination through Giemsa staining. Neutrophils were cultured in complete Roswell Park Memorial Institute (RPMI) medium, pre-incubated with or without rutin ($25{\mu}M$) for 45 minutes, and stimulated with phorbol 12-myristate 13-acetate (PMA). Then, the $TNF-{\alpha}$, NO and MPO productions were analyzed using enzyme-linked immunosorbent assay (ELISA), Griess Reagent, and MPO assay kits, respectively. Also, the viability of human neutrophils was assessed using tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and neutrophils were treated with various concentrations of rutin ($1-100{\mu}M$), after which MTT was appended and incubated at $37^{\circ}C$ for 4 hour. Results: Rutin at concentrations up to $100{\mu}M$ did not affect neutrophil viability during the 4-hour incubation period. Rutin significantly decreased the NO and $TNF-{\alpha}$ productions in human peripheral blood neutrophils compared to PMA-control cells (P < 0.001). Also, MPO activity was significantly reduced by rutin (P < 0.001). Conclusion: In this in vitro study, rutin had an anti-inflammatory effect due to its inhibiting NO and $TNF-{\alpha}$ productions, as well as MPO activity, in activated human neutrophils. Treatment with rutin may be considered as a therapeutic strategy for neutrophil-mediated inflammatory/autoimmune diseases.

• Journal of the Korean Society of Food Science and Nutrition
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• v.41 no.12
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• pp.1671-1676
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• 2012

In this study, the immuno-enhancing activity of seed extracts were studied on the macrophage cell lines. We examined the effect of nine seed extracts on nitric oxide (NO) production in RAW 264.7 cells and selected four highly-effective seed candidates (Fagopyrum esculentum, Taraxacum platycarpum, Impatiens balsamina, Helianthus annuus) for further immune-related studies. The effects of the four seed extracts on the production of immune-related cytokines in the RAW 264.7 macrophage cell line and proliferation of Molt-4 as a T cell line were investigated. The secretion of NO from the RAW 264.7 cells was increased up to 39 ${\mu}M$ by adding the seed extracts (25 ${\mu}g/mL$) compared to the control. Also, the secretion of tumor necrosis factor-alpha (TNF-${\alpha}$) was also increased up to 32 times by adding the seed extracts (25 ${\mu}g/mL$). Secretion of cytokines such as interleukin-1 beta (IL-$1{\beta}$), interleukin-6 (IL-6), and interleukin-10 (IL-10) was also increased and induced the proliferation of T cells compared to the control. In conclusion, these results suggest that four seed extracts provide beneficial immuno-enhancing effects for human health.

• Clinical and Experimental Reproductive Medicine
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• v.50 no.1
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• pp.44-52
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• 2023

Objective: The DNA integrity of spermatozoa that attach to fallopian tube (FT) cells is higher than spermatozoa that do not attach. FT epithelial cells can distinguish normal and abnormal sperm chromatin. This study investigated the effects of sperm with a high-DNA fragmentation index (DFI) from men with unexplained repeated implantation failure (RIF) on the Toll-like receptor (TLR) signaling pathway in human FT cells in vitro. Methods: Ten men with a RIF history and high-DFI and 10 healthy donors with low-DFI comprised the high-DFI (>30%) and control (<30%) groups, respectively. After fresh semen preparation, sperm were co-cultured with a human FT epithelial cell line (OE-E6/E7) for 24 hours. RNA was extracted from the cell line and the human innate and adaptive immune responses were tested using an RT2 profiler polymerase chain reaction (PCR) array. Results: The PCR array data showed significantly higher TLR-1, TLR-2, TLR-3, TLR-6, interleukin 1α (IL-1α), IL-1β, IL-6, IL-12, interferon α (IFN-α), IFN-β, tumor necrosis factor α (TNF-α), CXCL8, GM-CSF, G-CSF, CD14, ELK1, IRAK1, IRAK2, IRAK4, IRF1, IRF3, LY96, MAP2K3, MAP2K4, MAP3K7, MAP4K4, MAPK8, MAPK8IP3, MYD88, NFKB1, NFKB2, REL, TIRAP, and TRAF6 expression in the high-DFI group than in the control group. These factors are all involved in the TLR-MyD88 signaling pathway. Conclusion: The MyD88-dependent pathway through TLR-1, TLR-2, and TLR-6 activation may be one of the main inflammatory pathways activated by high-DFI sperm from men with RIF. Following activation of this pathway, epithelial cells produce inflammatory cytokines, resulting in neutrophil infiltration, activation, phagocytosis, neutrophil extracellular trap formation, and apoptosis.

• Development and Reproduction
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• v.15 no.2
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• pp.113-120
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• 2011

BCL-2 family essential proteins to play a pivotal role to perform in apoptosis signaling pathways and essential proteins for the regulation of cell death. BCL2L10 protein is a member of BCL-2 family and it regulates both anti-apoptotic and pro-apoptotic function of specific tissue or cell line. BCL2L10 of function and expression is not reported in ovary cell lines. In this study we reported that BCL2L10 were significant expression of KGN cell line. Ectopic expression of BCL2L10 induced cell death, and its cells killing effect was blocked by pan-caspase inhibitor of the Z-VAD-fmk. Ectopic expression of BCL2L10 protein led to the activation of caspase 9 and caspase 3, suggesting apoptotic cell death, and confocal microscopic analyses showed that BCL2L10 was partially localized in mitochondria. Thus, we provide a novel function of BCL2L10 in KGN cells, which was involved in the intrinsic cell death pathway.

• Molecules and Cells
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• v.41 no.9
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• pp.868-880
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• 2018

Pancreatic cancer (PC) is one of the most aggressive cancers presenting with high rates of invasion and metastasis, and unfavorable prognoses. The current study aims to investigate whether EZH2/miR-139-5p axis affects epithelial-mesenchymal transition (EMT) and lymph node metastasis (LNM) in PC, and the mechanism how EZH2 regulates miR-139-5p. Human PC and adjacent normal tissues were collected to determine expression of EZH2 and miR-139-5p, and their relationship with clinicopathological features of PC. Human PC cell line was selected, and treated with miR-139-5p mimics/inhibitors, EZH2 vector or shEZH2 in order to validate the regulation of EZH2-mediated miR-139-5p in PC cells. Dual-luciferase report gene assay and chromatin immunoprecipitation assay were employed to identify the relationship between miR-139-5p and EZH2. RT-qPCR and Western blot analysis were conducted to determine the expression of miR-139-5p, EZH2 and EMT-related markers and ZEB1/2. Tumor formation ability and in vitro cell activity were also analyzed. Highly-expressed EZH2 and poorly-expressed miR-139-5p were detected in PC tissues, and miR-139-5p and EZH2 expressions were associated with patients at Stage III/IV, with LNM and highly-differentiated tumors. EZH2 suppressed the expression of miR-139-5p through up-regulating Histone 3 Lysine 27 Trimethylation (H3K27me3). EMT, cell proliferation, migration and invasion were impeded, and tumor formation and LNM were reduced in PC cells transfected with miR-139-5p mimics and shEZH2. MiR-139-5p transcription is inhibited by EZH2 through up-regulating H3K27me3, thereby down-regulation of EZH2 and up-regulation of miR-139-5p impede EMT and LNM in PC. In addition, the EZH2/miR-139-5p axis presents as a promising therapeutic strategy for the treatment of PC.

• Journal of Microbiology and Biotechnology
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• v.9 no.1
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• pp.98-105
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• 1999

Intestinal bacteria comprise one-third of the contents of the large intestine in humans. Their interactions with the gastrointestinal immune system induce characteristic immunological responses which stimulate or suppress the host's defense system. RAW 264.7 murine cell line was used as a macrophage model to assess the effects of the exposure to the isolated human intestinal bacteria, Bacteroides, Bifidobacterium, Eubacterium, Streptococcus, and E. coli, on NO (nitric oxide), $H_2O_2$(hydrogen peroxide), and cytokines IL (interleukin)-6 and TNF (tumor necrosis factor)-a production. RAW 264.7 cells were cultured in the presence of heat-killed bacteria for 24 h at concentrations of 0-$50\mu$g/ml. Our results showed that Bacteroides and E. coli stimulated IL-6, TNF-$\alpha$, NO, and $H_2O_2$production at high levels even at $1\mu$g/ml, whereas Bifidobacterium, Eubacterium, and Streptococcus showed a low level of stimulation at $1\mu$g/ml, and a gradual increase as the cell concentration increased up to $50\mu$g/ml. This result suggests that gram-negative Bacteroides and E. coli are better able to stimulate macrophage than gram-positive Bifidobacterium, Streptococcus, and Eubacterium. The in vitro approaches employed here should be useful in further characterization of the effects of intestinal bacteria on gastrointestinal and systemic immunity.