• Journal of Microbiology and Biotechnology
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• v.34 no.4
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• pp.920-929
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• 2024

As a pivotal defensive line against multitudinous malignant tumors, natural killer (NK) cells exist in the tumor microenvironment (TME). RAD18 E3 Ubiquitin Protein Ligase (RAD18) has been reported to foster the malignant progression of multiple cancers, but its effect on NK function has not been mined. Here, the study was designed to mine the mechanism by which RAD18 regulates the killing effect of NK cells on colorectal cancer (CRC) cells. Expression of E2F Transcription Factor 7 (E2F7) and RAD18 in CRC tissues, their correlation, binding sites, and RAD18 enrichment pathway were analyzed by bioinformatics. Expression of E2F7 and RAD18 in cells was assayed by qRT-PCR and western blot. Dual-luciferase assay and chromatin immunoprecipitation (ChIP) assay verified the regulatory relationship between E2F7 and RAD18. CCK-8 assay was utilized to assay cell viability, colony formation assay to detect cell proliferation, lactate dehydrogenase (LDH) test to assay NK cell cytotoxicity, ELISA to assay levels of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), and immunofluorescence to detect expression of toxic molecules perforin and granzyme B. High expression of RAD18 and E2F7 was found in CRC tissues and cells. Silencing RAD18 could hamper the proliferation of CRC cells, foster viability and cytotoxicity of NK cells, and increase the secretion of GM-CSF, TNF-α, IFN-γ as well as the expression of perforin and granzyme B. Additionally, ChIP and dual-luciferase reporter assay ascertained the binding relationship between RAD18 promoter region and E2F7. E2F7 could activate the transcription of RAD18, and silencing RAD18 reversed the inhibitory effect of E2F7 overexpression on NK cell killing. This work clarified the inhibitory effect of the E2F7/RAD18 axis on NK cell killing in CRC, and proffered a new direction for immunotherapy of CRC in targeted immune microenvironment.

• Biomedical Science Letters
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• v.29 no.4
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• pp.371-375
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• 2023

Natural killer (NK) cells are innate immune cells and play important roles as the first immune cells to recognize and kill cancer. In patients with advanced and terminal cancer, NK cells are often inactivated, suggesting that NK cells may play important roles in cancer treatment. In particular, the proportion of NK cells among immune cells infiltrating tumor tissues is often low, which suggests that NK cells do not survive in tumor microenvironment (TME). In order to overcome these hurdles of NK cells in cancer treatment, it is critical to develop strategies that enhance the proliferation and cytolytic activity of NK cells. We applied Vemurafenib to NK cells and measured the degree of NK cell proliferation and functional activation. We obtained unexpected results of increased NK cell numbers and anti-tumor activity after Vemurafenib treatment. Although further investigation is required to uncover the detailed mechanisms, our results suggest that Vemurafenib is a promising candidate to increase the efficacy of cancer immunotherapy using NK cells.

• YAKHAK HOEJI
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• v.53 no.3
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• pp.125-129
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• 2009

H2-M3 (M3) is a unique antigen presenting molecule which provides N-formylated peptide to certain type of T cells. Previous observation indicated that NK cell activity is significantly diminished during listerial infection in $H2-M3^{-/-}$ mice. To explore the possibility that M3 expression directly effect on NK cell activity, we measured NK cell activity with or without stimulation of N-formylated peptide on antigen presenting cells. Results indicated that the expression of M3 is not directly influence on NK cell activity. Further study will be focused on the indirect effect of M3 on regulating NK cell activity.

• IMMUNE NETWORK
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• v.5 no.4
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• pp.247-251
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• 2005

Background: Natural killer (NK) cells are CD3 (-) CD14 (-) CD56 (+) lymphocytes. They play an important role in the body's innate immune response. They can induce spontaneous killing of cancer cells or virus-infected cells via the Fas/Fas ligand or the granzyme/perforin systems. The corticotropin-releasing hormone (CRH) is an important regulator for the body's stress response. It promotes proliferation and migration of various cancer cells through the CRH type 1 receptor under stress, and also inhibits NK or T cell activity. However, the relationship of CRH and NK cell migration to the target has not been confirmed. Herein, we study the effect of CRH on NK cell migration. Methods: We used the human NK cell line, NK-92MI, and tested the expression of CRH receptor type 1 on NK-92MI by RT-PCR. This was to examine the effect of CRH on tumor and NK cell migration, thus NK cells (NK-92MI) were incubated with or without CRH and then each CRH treated cell's migration ability compared to that of the CRH untreated group. Results: We confirmed that CRH receptor type 1 is expressed in NK-92MI. CRH can decrease NK cell migration in a time-/dose-dependent manner. Conclusion: These data suggest CRH can inhibit NK cell migration to target cells.

• Biomedical Science Letters
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• v.29 no.3
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• pp.178-183
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• 2023

Natural killer (NK) cells are innate cytotoxic lymphoid cells that actively prevent neoplastic development, growth, and metastatic dissemination in a process called cancer immunosurveillance. Regulation of the cytotoxic activity of NK cells relies on integrated interactions between inhibitory receptors and numerous activating receptors that act in tandem to eliminate tumor cells efficiently. Conventional chemotherapy is designed to produce an anti-proliferative or cytotoxic effect on early tumor cell division. Therapies designed to kill cancer cells and simultaneously maintain host anti-tumor immunity are attractive strategies for controlling tumor growth. Depending on the drug and dose used, several chemotherapeutic agents cause DNA damage and cancer cell death through apoptosis, immunogenic cell death, or other forms of non-killing (i.e., mitotic catastrophe, senescence, autophagy). Among stress-induced immunostimulatory proteins, changes in the expression levels of NK cell activating and inhibitory ligands and tumor cell death receptors play an important role in the detection and elimination by innate immune effectors including NK cells. Therefore, we will address how these cytotoxic lymphocytes sense and respond to high and low concentrations of drug-induced stress to the drug cisplatin, among the various types of drugs that contribute to their anticancer activity.

• IMMUNE NETWORK
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• v.7 no.3
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• pp.133-140
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• 2007

Background: It is well established that cross talk between natural killer (NK) cells and myeloid dendritic cells (DC) leads to NK cell activation and DC maturation. In the present study, we investigated whether type 1-polarized DC (DC1) matured in the presence of IFN-${\gamma}$ and type 2-polarized DC (DC2) matured in the presence of PGE2 can differentially activate NK cells. Methods: In order to generate DC, plastic adherent monocytes were cultured in RPMI 1640 containing GM-CSF and IL-4. At day 6, maturation was induced by culturing the cells for 2 days with cytokines or PGE2 in the presence or absence of LPS. Each population of DC was cocultured with NK cells for 24 h. The antigen expression on DC was analyzed by flow cytometry and cytokine production in culture supernatant was measured by ELISA or a bioassay for TNF-${\alpha}$ determination. NK cell-mediated lysis was determined using a standard 4h chromium release assay. Results: DC2, unlike DC1, had weak, if any, ability to induce NK cell activation as measured by IFN-${\gamma}$ production and cytolytic activity. DC2 were weakly stimulated by activated NK cells compared to DC1. In addition, IFN-${\gamma}$-primed mature DC appeared to be most resistant to active NK cell-mediated lysis even at a high NK cell/DC ratio. On the other hand, PGE2-primed DC were less resistant to feedback regulation by NK cells than IFN-${\gamma}$-primed mature DC. Finally, we showed that the differential effect of two types of DC population on NK cell activity is not due to differences in their ability to form conjugates with NK cells. Conclusion: These results suggest that different combinations of inflammatory mediators differentially affect the effector function of DC and, as a result, the function of NK cells, eventually leading to distinct levels of activation in adaptive immunity.

• IMMUNE NETWORK
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• v.4 no.4
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• pp.205-215
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• 2004

In the early host defense system, effector function of natural killer (NK) cells results in natural killing against target cells such as microbe-infected, malignant, and certain allogenic cells without prior stimulation. NK cell cytotoxicity is selectively regulated by homeostatic prevalence between a repertoire of both activating and inhibitory receptors, and the discrimination of untransformed cells is achieved by recognition of major histocompatibility complex (MHC) class I alleles through inhibitory signals. Although it is well known that the bipotential T/NK progenitors are derived from the common precusor, functional mechanisms in terms of the development of NK cells remain to be further investigated. NK cells are mainly involved in innate immunity, but recent studies have been reported that they also play a critical role in adaptive immune responses through interaction with dendritic cells (DC). This interaction will provide effector functions and development of NK cells, and elucidation of its precise mechanism may lead to therapeutic strategies for effective treatment of several immune diseases.

• Journal of Society of Preventive Korean Medicine
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• v.16 no.3
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• pp.139-153
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• 2012

Objectives : NK cells are spontaneously cytotoxic lymphocytes. These are not only important parts in the first line of defence against bacterial and viral infections of outside, but they may also play a critical role in chronic viral diseases. NK cells kill their targets spontaneously, without the need for prior sensitization and class I MHC restriction by the regulation of cytolytic functions and secretion of a variety of cytokines, such as interleukin-12(IL-12), MCP-1, IL-6, TNF-${\alpha}$, IFN-${\gamma}$. In addition, macrophage and NK cells cooperate through the production of cell mediates. These cooperation and modulation are one of major factors to prevent for evading immune surveillance of cancer. Hence, it could be assumed that if any candidate to enhance activities of macrophage and NK cell, it is considered as a potentially useful agents against cancer. Methods : In our study, to investigate effect of fermented soybean extracts by Lactic acid bacteria (SFE, soybean fermented extracts) work on intestinal immune cell to maintain general immune modulating and anti-cancer activity. We analyzed NK cytotoxicity assay and gene expressions of cytokine related with macrophage and NK cell activity. Results : In vitro experiment, SFE was verified as safety material for cell toxicicty to tumor cell strain without any toxicity of tumor growth inhibition and various cell strain. Effects of macrophage activity stimulating directly by SFE measured induced cytokine. The studies showed that IL-12 production by stimulation of SFE depended on concentration from 0.16mg/mL to 0.63mg/mL with non toxicity to cell, and it was the best activity at 0.63mg/mL. Besides, the effective concentration of SFE producing TNF-${\alpha}$ is similar to IL-12, but it was the best activity at 1.25mg/mL. The level of MCP-1, IL-6 and IFN-${\gamma}$ depended on concentration from 0.16mg/mL to 10mg/mL, IFN-${\gamma}$ showed the best activity at the effective concentration of 0.63mg/mL. With the result of NK cell activity measurement, the spleen cell of mouse injected SFE had 1.5 times higher killing effect than non injected cell. Conclusions : The result of this studies is that Soybean fermetated extracts(SFE) has possibility to immune aided material for the function not only inhibition of microbial infection to macrophage but also activity of adaption immune and cellular immune system.

• Parasites, Hosts and Diseases
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• v.29 no.3
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• pp.267-278
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• 1991

The natural killer (NK) cell activity of splenocytes and recycling capacity of NK cells were observed by combining the $^{51}Cr-release$ cytotoxicity assay and single cell cytotoxicity assay against YAC-1. The ICR mice were infected intranasally with Naegleria fewleri, that is a pathogenic free-living amoeba. The mice infected with $1{\times}10^5$ trophosoites showed mortality rate of 76.7% and mean survival time of 12. 9 days. The cytotoxic activity of NK cells in infected mice was significantly higher than that of non-infected mice during the period between 12 hours and day 3 after infection, and highest on day 1. The target-binding capacity of NK cells in infected mice was not different from that of non-infected ones. Maximal killing potential and maximal recycling capacity were remarkably increased in infected mice as compared with the control. The results obtained in this observation indicated that elevated NK cell activity in mice infected with N. fowieri was not due to target-binding capacity of NK cells but due to the increased activity of NK cells and increased recycling capacity of individual NK cells.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7965-7970
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• 2014

One of the goals of tumor immunotherapy is to generate immune cells with potent anti-tumor activity through in vitro techniques using peripheral blood collected from patients. However, cancer patients generally have poor immunological function. Thus using patient T cells, which have reduced in vitro proliferative capabilities and less tumor cell killing activity to generate lymphokine-activated killer (LAK) cells, fails to achieve optimal clinical efficacy. Interleukin-2 (IL-2) is a potent activating cytokine for both T cells and natural killer cells. Thus, this study aimed to identify optimal donors for allogeneic LAK cell immunotherapy based on single nucleotide polymorphisms (SNP) in the IL-2 and IL-2R genes. IL-2 and IL-2R SNPs were analyzed using HRM-PCR. LAK cells were derived from peripheral blood mononuclear cells by culturing with IL-2. The frequency and tumor-killing activity of LAK cells in each group were analyzed by flow cytometry and tumor cell killing assays, respectively. Regarding polymorphisms at IL-2-330 (rs2069762) T/G, LAK cells from GG donors had significantly greater proliferation, tumor-killing activity, and IFN-${\gamma}$ production than LAK cells from TT donors (P<0.05). Regarding polymorphisms at IL-2R rs2104286 A/G, LAK cell proliferation and tumor cell killing were significantly greater in LAK cells from AA donors than GG donors (P<0.05). These data suggest that either IL-2-330(rs2069762)T/G GG donors or IL-2R rs2104286 A/G AA donors are excellent candidates for allogeneic LAK cell immunotherapy.