• Journal of Acupuncture Research
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• v.34 no.1
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• pp.1-9
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• 2017

Objectives : We investigated the synergistic effects of bee venom (BV) and natural killer (NK) cells on B16F10 melanoma cell apoptosis mediated by IL-4. Methods : We performed a cell viability assay to determine whether BV can enhance the inhibitory effect of NK-92MI cells on the growth of B16F10 melanoma cells, and western blot analysis to detect changes in the expression of IL-4, $IL-4R{\alpha}$, and other apoptosis-related proteins. EMSA was performed to observe the activity of STAT6. To confirm that the inhibitory effect of BV and NK cells was mediated by IL-4, the above tests were repeated after IL-4 silencing by siRNA (50 nM). Results : B16F10 melanoma cells co-cultured with NK-92MI cells and simultaneously treated by BV ($5{\mu}g/ml$) showed a higher degree of proliferation inhibition than when treated by BV ($5{\mu}g/ml$) alone or co-cultured with NK-92MI cells alone. Expression of IL-4, $IL-4R{\alpha}$, and that of other pro-apoptotic proteins was also enhanced after co-culture with NK-92MI cells and simultaneous treatment with BV ($5{\mu}g/ml$). Furthermore, the expression of anti-apoptotic bcl-2 decreased, and the activity of STAT6, as well as the expression of STAT6 and p-STAT6 were enhanced. IL-4 silencing siRNA (50 nM) in B16F10 cells, the effects of BV treatment and NK-92MI co-culture were reversed. Conclusion : These results suggest that BV could be an effective alternative therapy for malignant melanoma by enhancing the cytotoxic and apoptotic effect of NK cells through an IL-4-mediated pathway.

• Journal of Microbiology and Biotechnology
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• v.27 no.6
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• pp.1204-1208
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• 2017

Natural killer (NK) cells have been reported to be dysfunctional in chronic hepatitis B (CHB) infection. However, the functional recovery of NK cells under antiviral therapeutic agents in CHB was not clearly understood. In this study, we investigated the phenotypic changes of NK(CD56+CD3-) cells in terms of their functional markers (CD16, NKG2A, NKG2D) during tenofovir therapy in CHB. The frequency of NK(CD56+CD3-) cells in CHB patients was significantly increased after 12 months of tenofovir therapy when compared with baseline. The expression levels of CD16+/CD56+CD3- and NKG2A+/CD56+CD3- cells were also affected by tenofovir treatment. In addition, there was a positive correlation between the proportion of NK(CD56+CD3-) cells and HBV DNA (log copies/ml) in CHB patients.

• Animal cells and systems
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• v.4 no.4
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• pp.341-345
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• 2000

It has been very difficult to develop and evaluate efficient fish vaccines because fish immune cells have not been properly characterized. In this study, we investigated the cell-mediated immunological properties of B- and T-like cells in Nile tilapia (Oreochromis nilotica). Surface immunoglobulin negative ($slg^{-}$) cell population proliferated in response to mammalian T-cell mitogens PHA and Con A, while surface immunoglobulin positive ($slg^{+}$) cells responded to the B-cell mitogen LPS. The slg$^{[-10]}$ cells from hemocyanin (HC)-immunized Tilapia, compared to the non-immunized control, reacted more to PHA than to Con A. Unexpectedly, antigen (Ag)-specific response was observed in both $slg^{-}$ and $slg^{-}$cells. Regardless of HC immunization, whole leukocytes from 8 head kidney of fish showed natural killer (NK)cell activity. Especially, NK cell activity was much higher in slg$^{[-10]}$ cells than in slg$^{+}$cells, indicating the possibility that fish NK cells were not at least associated with slg$^{+}$ cell population and not activated by Ag. Further understanding of functional fish immune cells will help to evaluate and develop effective vaccines for fishes and to monitor the course of therapy In infected fishes.hes.

• Journal of Acupuncture Research
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• v.33 no.2
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• pp.1-9
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• 2016

Objectives : I investigated whether snake venom can synergistically strengthen the cytotoxic effects of NK-92 cells, and enhance the inhibition of the growth of lung cancer cells including NCI-H358 through the induction of death receptor dependent extrinsic apoptosis. Methods : Snake venom toxin inhibited cell growth of NCI-H358 Cells and exerted non influence on NK-92 cell viability. Moreover, when they were co-cultured with NK cells and concomitantly treated with $4{\mu}g/m{\ell}$ of snake venom toxin, more influence was exerted on the inhibition of growth of NCI-H358 cells than BV or NK cell co-culture alone. Results : The expression of Fas, TNFR2 and DR3 and in NCI-H358 lung cancer cells was significantly increased by co-culture of NK-92 cells and treatment of $4{\mu}g/m{\ell}$ of snake venom toxin, compared to co-culture of NK-92 cells alone. Coincidentally, Bax, caspase-3 and caspase-8 - expressions of pro-apoptotic proteins in the extrinsic apoptosis pathway, demonstrated significant increase. However, in anti-apoptotic NF-${\kappa}B$ activities, activity of the signal molecule was significantly decreased by co-culture of NK-92 cells and treatment of $4{\mu}g/m{\ell}$ of snake venom toxin, compared to co-culture of NK-92 cells or snake venom toxin treated by NCIH358 alone. Meanwhile, in terms of NO generation, there is a significant increase, in co-culture of NK-92 cells with NCI-H358 cells as well as the co-culture of NK-92 cells and concomitant treatment of $4{\mu}g/m{\ell}$ of snake venom toxin. However, no synergistic increase of NO generation was shown in co-culture of NK-92 cells and treatment of $4{\mu}g/m{\ell}$ of snake venom toxin, compared to co-culture of NK-92 cells with NCI-H358 cells. Conclusion : Consequently, this data provides that snake venom toxin could be useful candidate compounds to suppress lung cancer growth along with the cytotoxic effect of NK-92 cells through extrinsic apoptosis.

• The Korean Journal of Physiology and Pharmacology
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• v.16 no.3
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• pp.167-174
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• 2012

Natural killer (NK) cells provide one of the initial barriers of cellular host defense against pathogens, in particular intracellular pathogens. Because bone marrow-derived hematopoietic stem cells (HSCs), lymphoid protenitors, can give rise to NK cells, NK ontogeny has been considered to be exclusively lymphoid. Here, we show that porcine c-$kit^+$ bone marrow cells (c-$kit^+$ BM cells) develop into NK cells in vitro in the presence of various cytokines [interleukin (IL)-2, IL-7, IL-15, IL-21, stem cell factor (SCF), and fms-like tyrosine kinase-3 ligand (FLT3L)]. Adding hydrocortisone (HDC) and stromal cells greatly increases the frequency of c-$kit^+$ BM cells that give rise to $CD2^+CD8^+$ NK cells. Also, intracellular levels of perforin, granzyme B, and NKG2D were determined by RT-PCR and western blotting analysis. It was found that of perforin, granzyme B, and NKG2D levels significantly were increased in cytokine-stimulated c-$kit^+$ BM cells than those of controls. And, we compared the ability of the cytotoxicity of $CD2^+CD8^+$ NK cells differentiated by cytokines from c-$kit^+$ BM cells against K562 target cells for 28 days. Cytokines-induced NK cells as effector cells were incubated with K562 cells as target in a ratio of 100 : 1 for 4 h once a week. In results, $CD2^+CD8^+$ NK cells induced by cytokines and stromal cells showed a significantly increased cytotoxicity 21 days later. Whereas, our results indicated that c-$kit^+$ BM cells not pretreated with cytokines have lower levels of cytotoxicity. Taken together, this study suggests that cytokines-induced NK cells from porcine c-$kit^+$ BM cells may be used as adoptive transfer therapy if the known obstacles to xenografting (e.g. immune and non-immune problems) were overcome in the future.

• Biomolecules & Therapeutics
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• v.22 no.2
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• pp.106-113
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• 2014

In the present study, we investigated anti-cancer effect of snake venom activated NK cells (NK-92MI) in lung cancer cell lines. We used snake venom ($4{\mu}g/ml$) treated NK-92MI cells to co-culture with lung cancer cells. There was a further decrease in cancer cell growth up to 65% and 70% in A549 and NCI-H460 cell lines respectively, whereas 30-40% was decreased in cancer cell growth by snake venom or NK-92MI alone treatment. We further found that the expression of various apoptotic proteins such as that Bax, and cleaved caspase-3 as well as the expression of various death receptor proteins like DR3, DR4 and Fas was also further increased. Moreover, consistent with cancer cell growth inhibition, the DNA binding activity of NF-${\kappa}B$ was also further inhibited after treatment of snake venom activated NK-92MI cells. Thus, the present data showed that activated NK cells could further inhibit lung cancer cell growth.

• Archives of Pharmacal Research
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• v.26 no.10
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• pp.861-867
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• 2003

Inhibitory effect of the lectins (KML-C) isolated from Korean mistletoe (KM; Viscum album coloratum) on tumor metastases produced by murine tumor cells (B16-BL6 melanoma, colon 26M3.1 carcinoma and L5178Y-ML25 lymphoma cells) was investigated in syngeneic mice. An intravenous (i.v.) administration of KML-C (20-50 ng/mouse) 2 days before tumor inoculation significantly inhibited lung metastases of both B16-BL6 and colon 26-M3.1 cells. The prophylactic effect of 50 ng/mouse of KML-C on lung metastasis was almost the same with that of 100 $\mu$ g/mouse of KM. Treatment with KML-C 1 day after tumor inoculation induced a significant inhibition of not only the experimental lung metastasis induced by B16-BL6 and colon 26M3.1 cells but also the liver and spleen metastasis of L5178Y-ML25 cells. Furthermore, multiple administration of KML-C given at 3 day-intervals after tumor inoculation led to a significant reduction of lung metastasis and suppression of the growth of B16-BL6 melanoma cells in a spontaneous metastasis model. In an assay for natural killer (NK) cell activity. i.v. administration of KML-C (50 ng/mouse) significantly augmented NK cytotoxicity against Yac-1 tumor cells 2 days after KML-C treatment. In addition, treatment with KML-C (50 ng/mouse) induced tumoricidal activity of peritoneal macrophages against B16-BL6 and 3LL cells. These results suggest that KML-C has an immunomodulating activity to enhance the host defense system against tumors, and that its prophylactic and therapeutic effect on tumor metastasis is associated with the activation of NK cells and macrophages.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.9
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• pp.4039-4044
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• 2014

Background: The aim of this study was to investigate the anti-cancer effects and mechanisms of immunochemotherapy of 5-fluorouracil (5-FU) and interleukin-2 (IL-2) on non-small cell lung cancer (NSCLC) A549 cells. Materials and Methods: In order to detect whether 5-FU+IL-2 could effectively inhibit tumor growth in vivo, we established an A549-bearing nude mouse model. The cytotoxicity of natural killer (NK) cells was evaluated using a standard chromium release assay. To evaluate the relevance of NK cells in 5-FU+IL-2-mediated tumor inhibitory effects, we depleted NK cells in A549-bearing mice by injecting anti-asialo-GM-1 antibodies. Effects of 5-FU+IL-2 on the expression and promoter activity of NKG2D ligands (MICA/MICB) in A549 cells in vitro were also assessed. Results: In A549-bearing nude mice, combination therapy significantly inhibited tumor growth in comparison with monotherapy with 5-FU or IL-2 and enhanced the recognition and lysis of tumor cells by NK cells. Further study of mechanisms showed that NK cells played a vital role in the anticancer immune response of 5-FU+IL-2 immunochemotherapy. In addition, the combination therapy synergistically stimulated the expression and promoter activity of MICA/MICB. Conclusions: 5-FU and IL-2 immunochemotherapy significantly inhibited tumor growth and activated NK cytotoxicity in vivo, and these effects were partly impaired after depleting NK cells in tumor-bearing mice. Combination treatment of 5-FU and IL-2 upregulated the expression and the promoter activity of MICA/MICB in A549 cells, which enhanced the recognition of A549 cells by NK cells. All of the data indicated that immunochemotherapy of 5-FU and IL-2 may provide a new treatment option for patients with lung cancer.

• IMMUNE NETWORK
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• v.8 no.2
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• pp.53-58
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• 2008

Background: Molecular mechanisms of natural killer (NK) cell development from hematopoietic stem cells (HSCs) have not been clearly elucidated, although the roles of some genes in NK cell development have been reported previously. Thus, searching for molecules and genes related NK cell developmental stage is important to understand the molecular events of NK cell development. Methods: From our previous SAGE data-base, Gpnmb (Glycoprotein non-metastatic melanoma protein B) was selected for further analysis. We confirmed the level of mRNA and protein of Gpnmb through RT-PCR, quantitative PCR, and FACS analysis. Then we performed cell-based ELISA and FACS analysis, to know whether there are some molecules which can bind to Gpnmb. Using neutralizing antibody, we blocked the interaction between NK cells and OP9 cells, and checked IFN-${\gamma}$ production by ELISA kit. Results: Gpnmb expression was elevated during in vitro developmental stage and bound to OP9 cells, but not to NK precursor cells. In addition, we confirmed that the levels of Gpnmb were increased at NK precursor stage in vivo. We confirmed syndecan4 as a candidate of Gpnmb's binding molecule. When the interaction between NK cells and OP9 cells were inhibited in vitro, IFN-${\gamma}$ production from NK cells were reduced. Conclusion: Based on these observations, it is concluded that Gpnmb has a potential role in NK cell development from HSCs.

• The Korea Journal of Herbology
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• v.23 no.4
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• pp.81-89
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• 2008

Objectives: In this study, the pharmacological effects of the ethylacetate extract of Ostericum koreanum(North Kangwhal; NK) on allergic inflammation were investigated in activated human mast cells. Methods: North Kangwhal was extracted with 80% methanol for 24 h, and then fractionated with ethylacetate(NK-EtOAc extract). HMC-1 cells, an human mast line, were pre-incubated with different concentrations of NK-EtOAc extract for 30 min, and then stimulated with PMA(50 nM/ml) and A23187($1{\mu}M/ml$) at indicated times. The cell toxicity was determined by MTT assay. The concentrations of prostaglandin E2(PGE2) and cytokines(TNF-${\alpha}$, IL-8) were measured by enzyme-linked immunosorbant assay. Results: NK-EtOAc extract($10{\sim}50{\mu}g/ml$) significantly inhibited the productions of $PGE_2$, TNF-${\alpha}$ and IL-8 in PMA/A23187-stimulated HMC-1 cells without cell toxicity($0{\sim}50{\mu}g/ml$). NK-EtOAc extract also inhibited PMA/A23187-induced phosphorylation of ERK1/2 MAPK and the NF-${\kappa}B$ p65 subunit translocation into the nuclear of HMC-1 cells. Conclusions: This study suggests that NK-EtOAc extract may have an anti-inflammatory property through suppressing the production of inflammatory mediators in activated mast cells and its molecular mechanism underlies the blocking of NF-${\kappa}B$ pathway.