Mechanism of antimicrobial-peptide cytotoxicity in mammalian cells

박수현 건국대학교 대학원 2020 국내석사

최근, 다중약물내성을 갖는 병원성 미생물의 지속적인 증가가 보건 산업에서 우려할 만한 수준으로 나타나고 있다. 이러한 가운데, 자연면역체계의 일원으로 광범위한 병원성 미생물들에 대해 강한 활성을 가지는 항균펩타이드(AMP)가 대두되고 있다. 돼지 항균 펩타이드인 프로테그린-1(Protegrin1; PG1)은 양전하를 띠는 작은 펩타이드(Cationic Antimicrobial Peptide; CAT)로써 양친성 β결합의 헤어핀 구조를 가지며, 전하간 상호작용에 의해 활성화된다. 항균펩타이드는 미생물의 종류에 크게 상관없이 작용하며, 빠른 박멸과정을 보이기 때문에 내성이 없어 기존의 항생제에 대한 유망한 대안이 될 수 있다. 따라서, 항균펩타이드를 치료제로 활용하기 위해서는 포유동물 세포에 미치는 영향을 보다 자세히 이해할 필요가 있다. 본 연구는 다양한 포유동물 세포에 대한 프로테그린-1의 세포 독성 효과를 분석하여 포유동물세포 내 프로테그린-1의 작용 메커니즘을 밝히고자 하였다. 먼저 재조합 프로테그린-1을 생산하여 배아 섬유 아세포(NIH-3T3), 망막 세포(661W), 배아 신장 세포(HEK293T), 신경 아세포종 세포(SH-SY5Y), 폐포 대 식세포(3D4/2) 및 호중구 세포(PMN)를 포함하는 다양한 유형의 포유동물 세포주에서 세포 독성을 평가했다. 다양한 포유동물 세포주에 프로테그린-1을 처치하여 세포독성을 살핀 결과, 세포 유형별로 세포독성 효과가 달리 나타났으며, 그 중 망막 뉴런 세포(661W) 및 호중구 세포(PMN)가 프로테그린-1에 의한 세포독성에 가장 현저하게 영향을 받았다. 세포 유형별로 나타나는 독성 차이를 규명하기 위하여, 우리는 원평광 이색성 분석(circular dichroism analysis)을 통하여 프로테그린-1의 2차구조 형태 변화를 살폈다. 그 결과, 포유동물세포 내에서 프로테그린-1의 β-병풍구조 형성을 확인하였으며, 망막뉴런 세포(661W)에서 프로테그린-1의 2차 구조형성율이 가장 높은 것을 확인하였다. 이로써, 프로테그린-1의 2차구조 형성 정도가 세포독성 차이와 밀접하게 관련되어 있음을 확인할 수 있었다. 또한 양전하의 정도가 다른 두 종류의 녹색 형광 단백질(+5-6 s-GFP, +15-17 s-GFP)을 이용하여 전하 차이에 따른 세포내 투과 정도를 분석하였다. 그 결과, 모든 세포에서 양전하가 더 높은 녹색 형광 단백질(+15-17 s-GFP)이 더 높게 투과되었으며, 그 중 망막세포(661W)에서 녹색 형광 단백질이 가장 높게 투과되었다. 이를 통해, 양전하 단백질의 투과 정도가 세포마다 다름을 확인할 수 있었으며, 양전하를 띠는 프로테그린-1에 의한 세포독성 차이와 관련성이 있음을 증명하였다. 추가적으로, 염소산나트륨을 처리하여 음이온성 설페이트화 된 프로테오글리칸 합성을 저해하였으며, 이를 통해 다양한 세포의 세포막 음전하가 감소되었을 때 나타나는 세포 독성 차이를 분석하였다. 그 결과 세포막의 음이온 특성이 저해될 때, 프로테그린-1에 의한 세포 독성 손상이 감소되어 세포의 생존율이 증가되는 것을 확인 할 수 있었다. 결론적으로, 다양한 포유동물 세포 유형에서 프로테그린-1의 세포독성을 관찰함으로써 프로테그린-1의 2차 구조 및 양전하 정도, 세포막의 음이온 정도가 포유동물 세포독성에 직접적으로 관여함을 증명하였다. 따라서, 본 연구는 항균펩타이드의 포유동물 세포독성 기전을 규명하였음에 의의가 있으며, 향후 항균펩타이드 기반 신약 개발 등에 유용한 결과로 활용될 수 있을 것이다. Porcine protegrin-1 (PG-1) is a broad-spectrum antimicrobial peptide (AMP) with potent antimicrobial activities. We produced recombinant PG-1 and evaluated its cytotoxicity toward various types of mammalian cell lines, including embryonic fibroblasts (NIH-3T3), retinal cells (661W), embryonic kidney cells (HEK293T), neuroblastoma cells (SH-SY5Y), alveolar macrophage cells (3D4/2), and neutrophils (PMN). The sensitivity of the different mammalian cells to cytotoxic damage induced by PG-1 differed significantly among the cell types, with retinal neuron cells (661W) and neutrophils (PMN) being the most significantly affected. A circular dichroism (CD) analysis showed there was a precise correlation between conformational changes in PG-1 and the magnitude of cytotoxicity among the various cell types. Subsequently, a green fluorescent protein (GFP) penetration assay using positively charged GFPs indicated there was a close correlation between the degree of penetration of charged GFP into cells and the magnitude of PG-1 cytotoxicity. Furthermore, we also showed that inhibition of the synthesis of anionic sulphated proteoglycans on the cell surface decreases the cytotoxic damage induced by PG-1 treatment. Taken together, the observed cytotoxicity of PG-1 towards different membrane surfaces is highly driven by the membrane’s anionic properties. Our results reveal a possible mechanism underlying cell-type dependent differences in cytotoxicity of AMPs, such as PG-1, toward mammalian cells.

Heat killed Lactobacillus acidophilus La205 enhances NK cell cytotoxicity through an increased expression of granulysin

박정규 단국대학교 대학원 2010 국내석사

Heat-killed lactic acid bacteria (LAB) were effective in immunomodulation, such as tumor necrosis factor-alpha (TNF-α) production, nitric oxide release, and increased phagocytic activity in macrophages. However, the functions of heat-killed LAB remain unclear in natural killer (NK) cells. This study is tested the effect of heat-killed Lactobacillus acidophilus La205 (La205) in NK cells to investigate whether the effects of heat-killed LAB on immune function. The results showed that the lysis of NK cells treated with heat-killed La205 at ratio of 1:50 is an approximately two-fold augmentation as compared to the non-treated group. To investigate the mechanism that heat-killed La205 induces in NK cytotoxicity, a CD107a assay was performed. The expression of CD107a implies that the degranulation of cytolytic granules was increased by heat-killed La205. In addition, heat-killed La205 markedly enhanced granulysin mRNA expression. These data demonstrate that granulysin could be a mediator of heat-killed La205 enhanced NK cell cytotoxicity through increased degranulation of cytolytic granules. These data concluded that heat-killed La205 is an activator of human NK cell cytotoxicity. 열처리한 유산균 (사균체)는 TNF-α 생산, 산화질소 분비와 macrophage의 phagocytic 능력들을 면역 조절하는데 효과적이다. 그러나, 사균체와 NK cell에 미치는 기능은 아직까지 알려지지 않았다. 이 연구에서는, 사균체가 면역기능에 미치는 효과를 알아보기 위해 열처리한 Lactobacillus acidophilus La205가 NK cell에 효과를 알아보았다. 열처리한 La205는 NK cell에 1:50으로 처리시 대조구에 비해 NK cytotoxicity가 2배 증가하였다. 열처리한 La205가 NK cytotoxicity를 증가시키는 메커니즘을 알아보기 위해 CD107a assay를 수행하였다. Cytolytic granules의 degranulation의 정도를 나타내는 CD107a의 발현이 열처리한 La205에 의해 증가하였다. 그에 더하여, 열처리한 La205는 granulysin의 mRNA의 발현을 증가시킨다. 이러한 연구 결과는 granulysin은 열처리한 La205에 의해 증가된 NK cytotoxicity의 mediator가 될 수 있음을 보여준다. 이러한 결과로 볼 때, 열처리한 La205는 인간의 NK cell cytotoxicity의 activator이다.

Citrus flavonoids의 세포 보호 효과에 대한 연구

박종민 연세대학교 대학원 2001 국내석사

Flavonids는 식물계에 널리 존재하고 사람들이 자주 섭취하는 diphenylpropanes 유도체로, 대략 4000여종의 flavonoids가 존재하는 것으로 알려져 있으며, 다양한 생리 활성을 가지고 있는 것으로 연구되고 있다. 본 연구에서는 활성산소종이 생성단계에 따라 그 반응성이나 특성이 다르다는 사실에 주목하여 각각의 활성산소종에 대한 flavonoids의 반응성을 측정하고 세포 보호효과와 암세포 성장 저해 효과를 측정하였다. 실험한 8종류의 flavonoids(naringin, naringenin, hesperidin, neohesperidin, hesperitin, rutin, quercetin, catechin)은 모두 항산화성을 나타내었으며, 특히 quercetin, rutin, hesperetin, catechin은 생체 내 활성산소종 제거 효소보다도 높은 효과를 나타내었다. 또한 활성산소종을 만들 수 있는 전이금속(Cu2^(+), Fe2^(+))을 제거하는 효과와 생체 내 산화효소인 xanthine oxidase의 activity를 저해하는 효과를 보였다. 활성산소종에 의한 세포(HIT-T15)의 cytotoxicity에 대해서도 flavonoids는 높은 보호 효과를 나타내었고, flavonoids 그 자체로 normal cell line(HIT-T15)에 대해서 보다, human breast cancer cell line (MCF-7)에 특이적으로 cytotoxicity를 보였다. 유사한 구조를 가지고 있는 flavonoids의 대한 이러한 연구는 그것들이 가지고 있는 생리적 작용을 분자 레벨에서 이해하는데 기초자료가 된다고 평가하였다. Flavonoids are diphenylpropanes that commonly occur in plants and are frequently components of the human diet. More than 4000 flavonoids have been found. Some flavonoids have been found to possess a lot of biological effect. These biological effects are believed to come form the antioxidant properties of the related flavonoids. Citrus Flavonoids were tested for free radical scavenging activities, protective effects for the cytotoxicity of reactive oxygen species on HIT-T15 cell and anticancer activity. All of these flavonoids have antioxidant activity. Especially quercetin, rutin, hesperetin and catechin have high activity. Treated to HIT-T15 cells, flavonoids showed significant protection against ROS cytotoxicity. Furthermore, Some flavonoids showed cytotoxicity on human breast cancer cell (MCF-7) but, flavonoids didn't show cytotoxicity on normal cell (HIT-T15). Therefore, these citrus flavonoids may have a potential usage as a natural and physiological antioxidant materials and biological resource for a lot of disease.

암세포에 대한 인삼, 플라보노이드 유도체 및 퀴놀린 유도체의 독성작용 기작에 관한 연구

송선옥 연세대학교 대학원 2000 국내박사

Cytotoxicity and Anti-Metastatic Potential of Garlic (Allium sativum) Extracts

알무나위르 경상대학교 대학원 2009 국내박사

Nowadays, the medicinal use of garlic is widespread and even more increasing. Among others there are people who prefer to use fresh garlic (either cloves itself or sliced) than cooked one. This holds also true for many Koreans particularly with a plate of meat and vegetables. The aims of the present study were to explore the toxic mechanism of fresh garlic and the anti-metastatic effect of boiled garlic. Its toxicological effect on INT-407 intestinal epithelial cells and anti-metastatic effect on A549 lung cancer cells was investigated. In the first experiment, the cytotoxicity mechanism of fresh garlic component on INT-407 intestinal epithelial cells was analyzed. A brief exposure (20 min) to fresh garlic extract but not boiled garlic extract induced a significant increase of cell death in a concentration dependent manner (37 ± 2 % cell death at 300 u'g/mL of FGE). The high M.W. (>10 kDa) but not the low M.W. (<10 kDa) contains cytotoxic components. Using SDS-PAGE, the proteins of FGE were separated and found the molecular weights of about 70, 45, 35, 23 and 10 kDa proteins are major components. These results suggest that a brief exposure to fresh garlic can induce cytotoxic injury on INT-407 intestinal epithelial cells. In the second experiment, proteinous components were isolated from the bulbs of fresh garlic (Allium sativum) using a serial process of extraction with distilled water, 30~80 % ammonium sulfate precipitations, and size exclusion chromatography (Sephadex G-75). Alliinase and lectin were identified by MALDI/TOF Mass Spectrometer. The alliinase-lectin complex and alliinase shows a potent cytotoxicity on INT-407 intestinal epithelial cells (LD50=83.43 u'g/mL, LD50=1 052 u'g/mL), but lectin have less cytotoxicity. This result suggests that the proteinous cytotoxic component in fresh garlic is alliinase-lectin complex. In the third experiment, the anti-metastatic effect of boiled garlic extract (BGE) on A549 lung cancer cells was examined. Using scratch wound assay, agarose drop migration assay as well as colony forming assay, BGE inhibits the migration of A549 cell at 300 u'g/mL. In addition, the mechanism of migration inhibitory effect of boiled garlic was confirmed by Western blotting analysis. Interestingly, BGE inhibits migration A549 cell by decreasing the level of FAK and its phosphorylation (P-Y397) from 3 hr after the treatment. Taken together, the present finding demonstrates a novel anti-metastatic effect of garlic and suggests a certain garlic-derived compounds of BGE may be a potential agent for inhibiting migration by the modulation of FAK in cancer cells.

노루궁뎅이버섯 및 노란다발버섯의 細胞毒性 成分硏究

노형준 성균관대학교 일반대학원 2012 국내박사

Cytotoxic Constituents of Hericium erinaceum and Naematoloma fasciculare As a part of our continuing search for bioactive constituents from Korean medicinal and poisonous mushrooms, we investigated methanol extracts of the Hericium erinaceum and Naematoloma fasciculare by bioactivity-guided fractionation and isolation of the methanolic extracts of above two mushrooms, two new phenolic compounds (H-9, H-11), five known hericenones (H-1, H-4, H-5, H-7, H-8), a erinacerin B (H-3), a erinacerin A(H-10), a 3,4-dihydro-5-methoxy-2-methyl-2-(4'-methyl-2'-oxo-3'-pentenyl)-9(7H)-oxo-2H-furo[3,4-h]benzopyran (H-6) and two sterols (H-2, H-12) were obtained from the n-hexane-soluble fractions and CH2Cl2-soluble fractions from Hericium erinaceum. Their structures were determined to be hericenone D (H-1), ergosterol proxide; (22E,24R)-5α,8β-epidioxyergosta-6,22-dien-3β-ol (H-2), erinacerin B (H-3), hericenone A; 6-[(2&#61602;E)-3&#61602;,7&#61602;-dimethyl-5&#61602;-oxo-2&#61602;,6&#61602;-octadienyl]-7-hydroxy-5-methoxyphthalide (H-4), hericenone E (H-5), 3,4-dihydro-5-methoxy-2-methyl-2-(4'-methyl-2'-oxo-3'-pentenyl)-9(7H)-oxo-2H-furo[3,4-h]benzopyran (H-6), hericenone J (H-7), hericenone F; 8-formyl-5-methoxy-2-methyl-2-(4&#61602;-methyl-2&#61602;-oxo-3&#61602;-pentenyl)-7-chromanylmethyl palmitate (H-8), hericerin A (H-9), erinacerin A (H-10), hericenone B1 (H-11) and cerevisterol; (22E,24R)-ergosta-7,22-dien-3β,5α,6β-triol (H-12) using spectroscopic data, including 1D and 2D NMR. Cytotoxic activities of the isolated compounds (H-1 &#61485; H-12) were tested for their cytotoxicity against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, HCT15) in vitro using the sulforhodamine B bioassay. H-2, H-6, H-9 and H-10 exhibited moderate cytotoxic activity against A549, SK-OV-3, SK-MEL-2, and HCT15 cells (IC50 (H-2): 17.43, 16.81, 10.16 and 17.79 μM and IC50 (H-6): 17.44, 11.08, 13.24 and 15.63 μM and IC50 (H-9): 20.51, 8.93, 3.11, 19.46 μM and IC50 (H-10): 10.61, 11.14, 7.66 and 13.84 μM, respectively), and H-11, possessing a phenolic ring and ketone in the side chain, exhibited significant cytotoxic activity against the A549, SK-OV-3, SK-MEL-2, and HCT15 cell lines (IC50 (H-11): 2.63, 3.08, 1.87, and 2.90 μM, while the other compounds showed little cytotoxicity against the tested cell lines (IC50 > 30 μM). Two new lanostane triterpenoids (N-1 &#61485; N-2) and six known triterpenes (N-3 &#61485; N-8) were isolated from the CHCl3-soluble fraction from Naematoloma fasciculare. The structures were established to be fasciculol J (N-1), fasciculol K (N-2), fasciculol G (N-3), fasciculol B ; 2&#61537;,3&#61538;,12&#61537;,24R,25-pentahydroxylanosta-8-ene (N-4), fasciculic acid B (N-5), fasciculol C; 2&#61537;,3&#61538;,12&#61537;,21,24R,25-hexahydroxylanosta-8-ene (N-6), fasciculol H (N-7), fasciculol I (N-8) by using spectroscopic data, including 1D and 2D NMR. Among these isolates, compounds N-1 and N-2 were newly isolated from the nature. The cytotoxicities of the isolated compounds (N-1 &#61485; N-8) were tested for their cytotoxicity against four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, HCT15) in vitro using the sulforhodamine B bioassay. N-4 and N-6, exhibited significant cytotoxic activity against the A549, SK-OV-3, SK-MEL-2, and HCT15 cell lines (IC50 (N-4): 7.85, 8.53, 5.17, and 8.22 μM, and IC50 (N-6): 2.37, 2.82, 2.29, and 3.06 μM, respectively). This dissertation describes isolation, structural elucidation and cytotoxic activities of 20 compounds including two lanostane triterpenoids, two new phenolic compounds, six known triterpenes, five known hericenones, one erinacerin B, one erinacerin A, one 3,4-dihydro-5-methoxy-2-methyl-2-(4'-methyl-2'-oxo-3'-pentenyl)-9(7H)-oxo-2H-furo[3,4-h]benzopyran and two known sterols isolated from the fruit body of Hericium erinaceum and Naematoloma fasciculare. 7 of these compounds showed cytotoxicity against tested human cancer cell line. Keyword : Hericium erinaceum, Naematoloma fasciculare, structural-elucidation, cytotoxicity

Psammaplin A 유도체의 합성과 항암 활성에 관한 연구

김철우 서울대학교 대학원 2013 국내석사

Psammaplin A는 1987년 Psammaplysilla sponge등에서 처음 분리된 이 후 지속적인 후속연구 결과 antibacterial activity, 암세포에 대한 cyto-toxicity등 여러 가지 생리 활성을 가지고 있는 것으로 알려져 있다. 본 연구진은 Structure-Activity Relationship(SAR) 연구 결과 Psammaplin A보다 tetramethylated Psammaplin A 유도체가 약 10배 더 높은 cytotoxicity를 가지고 있다는 사실에 근거하여 oxime homodimer, amine homodimer, oxime heterodimer의 세 분류로 나누어 총 40종 이상의 유도체를 합성하였고, 이들 유도체의 A549 cell과 HTC116 cell 세포주에 대한 항암활성을 수행한 바 있다. 그 결과, oxime homodimer 계열과 aromatic ring의 분자단에 지용성이 큰 작용기를 포함하는 유도체에서 높은 cytotoxicity를 나타냄을 확인할 수 있었다. 그리하여 본 연구자는 더 높은 cytotoxicity를 나타내는 유도체를 도출하고자 다양한 할로겐으로 치환된 dimer이거나, oxime 부분에 다양한 alkyl chain을 가지는 dimer이거나, 또는 aromatic ring의 지용성을 극대화한 유도체를 설계하여 총 10종의 Psammaplin A 유도체를 합성하였다. 유도체의 합성은 본 연구실에서 개발하여 학계에 보고한 ethyl acetoacetate의 α-nitrosation 방법으로 진행하였다. 이렇게 합성된 유도체들은 6종의 암세포를 이용하여 항암활성을 측정한 결과, Psammaplin A 자체보다 항암활성이 우수한 5종의 유도체를 도출할 수 있었다. 이상의 결과는 향후 새로운 항암제의 개발에 응용될 수 있을 것으로 예상된다. In 1987, Psammaplin A isolated from Psammaplysilla sponge has been studied continually. As a result, Psammaplin A is known to have cytotoxicity to cancer cell. At 2012, we performed cytotoxicity tests of the 40 kinds of Psammaplin A derivative to A549 cell and HTC116 cell based on the result that Psammaplin A derivative has a cytotoxicity 10 times larger than Psammaplin A. We found that four Psammaplin A derivatives have effective cytotoxicity to cancer cell. Based on the result, I synthesized 10 kinds of Psammaplin A derivatives through modification of functional moiety for construction of focused library. Psammaplin derivatives for construction of focused library were tested to six kinds of cancer cell. I analyzed the result of cytotoxicity test.

Regulation of natural cytotoxicity receptor (NCR) expression on NK cells

김형란 Graduate School, Yonsei University 2007 국내박사

A number of surface receptors expressed on NK cells are related to the regulation of NK cell activity and characterized by either inhibitory or activating properties. Activating receptors usually have short cytoplasmic tails and transduce signals by associating with molecules containing Immunoreceptor Tyrosine-based Activating Motifs (ITAM). Natural cytotoxicity receptors (NCRs) are one major family of activating receptors involved in NK cytotoxicity, and are found only on NK cells. The three family members are NKp46 (NCR1), NKp44 (NCR2) and NKp30 (NCR3). Their surface density might vary with the activation state of NK cells, and the density may directly correlate with their natural cytotoxicity.In this study, we investigated the regulation of NCR expression on NK cells and the factors which affect it. We produced stable cell lines expressing full-length NCRs and investigated the change in expression after PMA treatment using flow cytometry, RT-PCR and immunoblotting methods. Expression of NKp30 and NKp46 on Jurkat T cell transfectants appeared to increase by PMA treatment until 8 hr after PMA treatment, but gradually decreased afterward to less than pre-treatment levels. Parallel to surface expression of NCRs, total NCR protein expression also appeared to fluctuate after PMA treatment, but expression of mRNA transcripts was not significantly affected. Experiments with mutant NCR-expressing stable cell lines demonstrated that 288Ser might be critical for NKp46 expression.In primary NK cells, most cytokines such as IL-2, IL-8, IL-12, IL-15, IL-18, IFN-α1 and IFN-α2b did not appear to significantly alter NCR expression. PD98059, PD150606 and Lactacystin also did not induce any notable changes, suggesting that the MAP kinase and proteosome pathways might not be involved in the regulation of NCR expression. Interestingly, however, PMA slightly down-regulated NKp46 expression on primary NK cells. PMA is a well-known PKC activator. Furthermore, although other PKC inhibitors did not induce or suppress NCR expression, Gö6983, an inhibitor of PKC α, β, γ, δ and ζ, induced a remarkable increase of NCR expression on NK cells. Finally, we show that up-regulation of NCR on NK cells by Gö6983 caused an increase in NK cytotoxicity against hepatocellular carcinoma cell lines (HCCs) and HeLa presumably by increasing granule release.In conclusion, NCR expression is down-regulated by PMA, a PKC activator, and upregulated by Gö6983, a PKC inhibitor. As a consequence, NK cytotoxicity against HCCs and HeLa appeared to greatly increase after Gö6983 treatment, but slightly decrease after PMA treatment. This suggests that a specific PKC inhibitor, such as Gö6983, could be utilized to enhance NK cytotoxicity and consequently increase host tumor immunity by upregulation of NCR expression. 자연살 세포(Natural Killer cells; NK 세포)는 종양세포 및 바이러스에 감염된 숙주세포를 제거하는 기능을 수행하며 부적절한 골수 이식을 거부하고 수상돌기세포를 통해서 T 세포의 면역반응을 조절하는 등 생체 내 면역계의 방어 기작의 제 1선에서 작용하는 매우 중요한 행동세포이다. 자연살 세포는 표면에 활성화 수용체(activating receptor)와 억제 수용체(inhibitory receptor)로 구분되는 여러 수용체들을 발현하고 있는데, 활성화 수용체 중에서도 자연 세포독성 수용체(Natural Cytotoxicity Receptors; NCRs)가 자연살 세포의 세포독성을 매개하는 주된 수용체이다. NKp46 (NCR1), NKp44 (NCR2) 그리고 NKp30 (NCR3)으로 구성되는 이 자연 세포독성 수용체들은 자연살 세포에서만 발현되고 그 표면 밀집도는 자연살 세포의 세포독성과 직접적으로 연관된다.본 연구는 자연살 세포에서의 자연 세포독성 수용체의 발현에 영향을 미치는 요인을 찾고 그 요소들이 어떻게 수용체의 발현을 조절하는지 알아보고자 하였다. Jurkat T 세포주를 사용하여 자연 세포독성 수용체를 발현하는 세포주를 구축하여 PMA 자극에 의해 변화하는 수용체의 발현을 유세포 분석과 RT-PCR과 immunoblotting으로 확인하였다. PMA에 의해서 NKp30과 NKp46의 발현이 8시간 정도까지 증가하다가 점차적으로 줄어들었으며 mRNA 양에서는 별 다른 변화를 관찰 할 수 없었다. 그에 비해 이들 자연 세포독성 수용체의 총 단백질량은 표면 발현과 비슷한 경향을 보였고 이러한 현상에 NKp46의 288번 serine이 중요한 역할을 하고 있음을 밝혀내었다.실제 자연살 세포에서의 자연 세포독성 수용체 발현에 영향을 미치는 인자들을 찾아내기 위해 기존에 면역계를 활성화 시킨다고 알려져 있던 여러 cytokine들을 탐색해 보았으나 크게 영향을 미치는 요소를 찾지 못했고, MEK 억제제인 PD98059, calpain 억제제인 PD150606과 proteosome 억제제인 Lactacystin도 자연 세포독성 수용체의 발현에 별 영향을 미치지 않는 것으로 나타났다. PMA 자극에 의해서 실제 자연살 세포에서는 NKp46의 발현이 다소 줄어들었다. PMA가 대표적인 PKC 활성제이므로 PKC 억제제가 자연 세포독성 수용체의 발현에 미치는 영향을 살펴보았다. 대부분의 PKC 억제제가 수용체의 발현에 아무런 영향을 미치지 못했는데, PKC α, β, γ, δ와 ζ를 억제하는 Gö6983에 의해서 NKp30과 NKp46의 발현이 현저히 증가하였다. Gö6983은 NKp30과 NKp46의 발현을 transcriptional level에서 조절함을 확인하였고 이렇게 증가된 수용체의 발현이 HepG2, Hep3B와 HeLa 암 세포주에 대한 자연살 세포의 세포독성의 증가에까지 영향을 미치는 것을 알 수 있었다.결론적으로, PMA에 의해서 다소 감소하고 Gö6983에 의해서 크게 증가하는 자연 세포독성 수용체의 발현이 자연살 세포의 세포독성을 조절하고, 따라서 이들 약물은 생체 내 면역계 활성에도 영향을 미칠 수 있을 것이라 사료된다.

Distribution and Correlation Between NK Cell Interferon γ Production and Cytotoxicity

임상은 성균관대학교 일반대학원 2018 국내석사

Background: The purpose of this study was to determine the distribution of interferon gamma level as a marker of natural killer cell activity (NKA-IFNγ) among subjects participating in a medical checkup program in Korea and to analyze the relationship between NKA-IFNγ and age, sex, body mass index (BMI), and smoking in a healthy Korean population. In addition, we analyzed NKA-IFNγ between healthy Koreans and patients with hypertension or diabetes mellitus. We also studied the association between NKA-IFNγ and various NK cell characteristics by measuring receptor expression and the correlation between NKA-IFNγ and NK cell cytotoxicity against K562 cancer cells. Materials and Methods: From July 2015 to December 2016, the levels of IFN-γ secreted after NK cell stimulation with PROMOCA™ were assayed in serum samples of 1998 healthy Koreans (1107 males and 891 females) using the NK vue kit (AT-Gen, Sungnam, Korea). Additionally, we performed multicolor flow cytometry assays to analyze the expression of various NK cell receptors (CD16, NKG2A, NKG2C, NKG2D, CD57, DNAM-1, CD8a, CD62L, NKp30, and NKp46) on both CD3-/CD56dim and CD3-/CD56bright NK cells in whole-blood samples from 119 healthy donors. The expression of these receptors was compared according to the level of NKA-IFNγ (< 100 pg/mL, n = 13, 100 to 250 pg/mL, n = 9, 250 to 500 pg/mL, n = 11, and > 500 pg/mL, n = 86). Flow cytometric NK cell cytotoxicity assays against K562 cells were performed with peripheral blood mononuclear cells from 40 individuals and the results were compared according to NKA-IFNγ. Results: The median levels of NKA-IFNγ were 1,340.05 pg/mL, 1,542.95 pg/mL, and 1,476.15 pg/mL for the total subjects, males, and females, respectively. Median levels of NKA-IFNγ were significantly higher in men than in women (p<0.0001). NKA-IFNγ tended to increase with increased BMI in a healthy Korean population (p for trend=0.000), but not with increased smoking (p for trend=0.558). There was no significant difference in NKA-IFNγ between a healthy population and patients with DM or HTN (p for trend=0.208, and 0.681, respectively). No correlation was found between the expression of NK cell receptors (CD16, NKG2A, NKG2C, NKG2D, CD57, DNAM-1, CD8a, CD62L, NKp30, and NKp46) and NKA-IFNγ (p = 0.519, p = 0.831, p = 0.592, p =0 220, p = 0.941, p = 0.056, p = 0.918, p = 0.596, p = 0.477, and p = 0.378, respectively). In addition, NK cell cytotoxicity was not correlated with NKA-IFNγ (p = 0.265). Conclusion: In the present study, the distribution of NKA-IFNγ was established in a Korean population. We observed no correlation between NK cell receptor expression and NKA-IFNγ, and also no correlation between NKA-IFNγ and NK cytotoxicity against K562 cancer cells. Therefore, foraccurate assessment of NK cell function in a medical checkup program, it is necessary to measure NKA-IFNγ in addition to other complementary tests including NK cytotoxicity.

Evaluation of IL-1β production and cytotoxicity induced by different forms of aluminum oxide particles in human monocytic THP-1 cells

김병일 Graduate School, Korea University 2011 국내석사

As the aluminum oxide (Al2O3) is relatively non-toxic and fairly chemically inert, it is widely used as a filter, catalyst support, and abrasive. However, the steady increase in production and usage of Al2O3 has raised the public concerns over the unpredictable health impacts. The biological effects of Al2O3 are considered to differ according to size and shape. Therefore, the association between the characteristics of Al2O3 and their safety needs to be evaluated. In this study, mechanisms of IL-1β production and cytotoxicity in macrophage-like THP-1 cells was investigated in response to the three different forms of Al2O3 particles (i.e., N-Al2O3 (<30 nm), S-Al2O3 (2~4 x 100~800 nm), L-Al2O3 (2~4 x 2800 nm)). It was demonstrated that Al2O3 induced different levels of IL-1β production and cytotoxicity according to its particle forms. Especially, it was found that S-Al2O3 induced higher levels of IL-1β production and cytotoxicity. It was also revealed that Al2O3-induced IL-1β production was dependent on the lysosomal destabilization, subsequent release of cathepsin B, and generation of intracellular reactive oxygen species (ROS). In addition, the cell death induced by Al2O3 was closely related with cathepsin B leakage, which was more likely to be necrosis than apoptosis. All these results have made it possible to propose the mechanisms of IL-1β production and cytotoxicity in the presence of different forms of Al2O3. The current finding might support the development of safe forms of Al2O3.