• Title/Summary/Keyword: As 4.1 cell line

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Immunotherapeutic Effects of CTLA4Ig Fusion Protein on Murine EAE and GVHD (마우스 EAE, GVHD 질환에서 CTLA4Ig 융합단백의 면역치료 효과)

  • Jang, Seong-Ok;Hong, Soo-Jong;Cho, Hoon-Sik;Chung, Yong-Hoon
    • IMMUNE NETWORK
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    • v.3 no.4
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    • pp.302-309
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    • 2003
  • Background: CTLA4 (CD152), which is expressed on the surface of T cells following activation, has a much higher affinity for B7 molecules comparing to CD28, and is a negative regulator of T cell activation. In contrast to stimulating and agonistic capabilities of monoclonal antibodies specific to CTLA-4, CTLA4Ig fusion protein appears to act as CD28 antagonist and inhibits in vitro and in vivo T cell priming in variety of immunological conditions. We've set out to confirm whether inhibition of the CD28-B7 costimulatory response using a soluble form of human CTLA4Ig fusion protein would lead to persistent inhibition of alloreactive T cell activation. Methods: We have used CHO-$dhfr^-$ cell-line to produce CTLA4Ig fusion protein. After serum free culture of transfected cell line we purified this recombinant molecule by using protein A column. To confirm characterization of fusion protein, we carried out a series of Western blot, SDS-PAGE and silver staining analyses. We have also investigated the efficacy of CTLA4Ig in vitro such as mixed lymphocyte reaction (MLR) & cytotoxic T lymphocyte (CTL) response and in vivo such as experimental autoimmune encephalomyelitis (EAE), graft versus host disease (GVHD) and skin-graft whether this fusion protein could inhibit alloreactive T cell activation and lead to immunosuppression of activated T cell. Results: In vitro assay, CTLA4Ig fusion protein inhibited immune response in T cell-specific manner: 1) Human CTLA4Ig inhibited allogeneic stimulation in murine MLR; 2) CTLA4Ig prevented the specific killing activity of CTL. In vivo assay, human CTLA4Ig revealed the capacities to induce alloantigen-specific hyporesponsiveness in mouse model: 1) GVHD was efficiently blocked by dose-dependent manner; 2) Clinical score of EAE was significantly decreased compared to nomal control; 3) The time of skin-graft rejection was not different between CTLA4Ig treated and control group. Conclusion: Human CTLA4Ig suppress the T cell-mediated immune response and efficiently inhibit the EAE, GVHD in mouse model. The mechanism of T cell suppression by human CTLA4Ig fusion protein may be originated from the suppression of activity of cytotoxic T cell. Human CTLA4Ig could not suppress the rejection in mouse skin-graft, this finding suggests that other mechanism except the suppression of cytotoxic T cell may exist on the suppression of graft rejection.

Assessment of Biomarkers in Acetaminophen-Induced Hepatic Toxicity by siRNA

  • Kang, Jin-Seok;Yum, Young-Na;Kim, Joo-Hwan;Park, Sue-Nie
    • Biomolecules & Therapeutics
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    • v.17 no.4
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    • pp.438-445
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    • 2009
  • We investigated global gene expression from both mouse liver and mouse hepatic cell lines treated with acetaminophen (APAP) in order to compare in vivo and in vitro profiles and to assess the feasibility of the two systems. During our analyses of gene expression profiles, we picked up several down-regulated genes, such as the cytochrome P450 family 51 (Cyp51), sulfotransferase family cytosolic 1C member 2 (Sult1c2), 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (Hmgcs1), and several genes that were up-regulated by APAP, such as growth arrest and DNA-damage-inducible 45 alpha (Gadd45a), transformation related protein 53 inducible nuclear protein 1 (Trp53inp1) and zinc finger protein 688 (Zfp688). For validation of gene function, synthesized short interfering RNAs (siRNAs) for these genes were transfected in a mouse hepatic cell line, BNL CL.2, for investigation of cell viability and mRNA expression level. We found that siRNA transfection of these genes induced down-regulation of respective mRNA expression and decreased cell viability. siRNA transfection for Cyp51 and others induced morphological alterations, such as membrane thickening and nuclear condensation. Taken together, siRNA transfection of these six genes decreased cell viability and induced alteration in cellular morphology, along with effective inhibition of respective mRNA, suggesting that these genes could be associated with APAP-induced toxicity. Furthermore, these genes may be used in the investigation of hepatotoxicity, for better understanding of its mechanism.

Effect of Ailanthi Cortex on the Apoptosis and Cell Cycle of HL-60 Leukemia Cell Line (HL-60 백혈병 세포의 세포고사에 미치는 저근백피의 효과)

  • Jeong Young Mok;Park Sin Ki;Lee Jun;Kim Young Mok;Yun Yong Gab;Kim Won Sin;Han Dong Min;An Won Gun;Yoon Yoo Sik;Jeon Byung Hun
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.17 no.4
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    • pp.914-922
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    • 2003
  • Ailanthus altissima has been used to settle an upset stomach, to alleviate a fever, and as an insecticide. We reported that the water extract of A. altissima induced apoptotic cell death in HL-60 human leukemia cell line. Here, we showed the dose-dependent inhibitions of cell viability by the extract, as measured by cell morphology. The cell cycle control genes are considered to play important roles in tumorigenesis. The purpose of the present study is also to investigate the effect of A. altissima on cell cycle progression and its molecular mechanism in the cells. The level of p21 protein was increased after treatment of the extract, whereas both Bcl-2 and Bax protein levels were not changed. These results suggest that A. altissima induces apoptotic cell death via p21-dependent signaling pathway in HL-60 human leukemia cell line which delete wild type p53. G1 checkpoin related gene products tested (cyclin D3, cyclin dependent kinase 4, retinoblastoma, E2F1) were decreased in their protein levels in a dose-dependent manner after treatment of the extract. Taken together, these results indicate that the increase of apoptotic cell death by A. altissima may be due to the inhibition of cell cycle in HL-60 human leukemia cell line

Autophagy-enhancing and neuroprotective effects of Wonji-Gobon mixture (WGM) in a Parkinson's disease mouse model

  • Lee, Jin-Wook;Kwak, Jin-Young;Koh, Young-Mee;Ahn, Taek-Won
    • Journal of Applied Biological Chemistry
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    • v.61 no.4
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    • pp.341-349
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    • 2018
  • The aim of this study was to evaluate autophagy-enhancing and neuroprotective effects of Wonji-Gobon mixture (WGM), a traditional Chinese prescription medication, in Parkinson's disease (PD) mouse models. Our investigation found that WGM increased the expression of both Beclin1 and LC3b-II proteins as measured with western blot in the BV2 cell line; both proteins play a role in autophagy. WGM also increased the autophagy expression as measured by fluorescence-activated cell-sorting analysis in the BV2 cell line. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD models, WGM significantly increased the amount of dopamine in a striatum-substantia nigra suspension, produced notable results in the forced swim test, and increased serotonin as measured by high-performance liquid chromatography analysis; these results are indicative of neuroprotective effects. In summary, our findings indicate that WGM treatment has neuroprotective effects that are partially mediated by autophagy enhancement.

Selective Inhibition of Bicyclic Tetrapeptide Histone Deacetylase Inhibitor on HDAC4 and K562 Leukemia Cell

  • Li, Xiao-Hui;Huang, Mei-Ling;Wang, Shi-Miao;Wang, Qing
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.12
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    • pp.7095-7100
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    • 2013
  • Histone deacetylase (HDAC) inhibitors of cyclic peptide have been proved to be the most complex but the most stable and relative efficient inhibitors because of their large cap region. In this paper, a series of studies were carried out to evaluate the efficacy of synthetic bicyclic tetrapeptide inhibitors 1-5 containing hydroxamic acid referring molecular docking, anti-proliferation, morphology and apoptosis. Docking analysis, together with enzyme inhibitory results, verified the selective capability of inhibitor 4 to HDAC4, which might closely related to haematological tumorigenesis, with Phe227, Asp115, Pro32, His198 and Ser114 participating into hydrophobic interactions and Van der Waals force which was familiar with former study. Moreover, inhibitor 4 inhibited K562 cell line at the $IC_{50}$ value of 1.22 ${\mu}M$ which was 51-67 times more efficient than that for U937 and HL60 cell lines. Inhibitor 4 exhibited the cell cycle-arrested capability to leukemia at S phase or G2/M phase as well as apoptosis-induced ability in different degrees. Finally, we considered that bicyclic tetrapeptide inhibitors were promising inhibitors used in cancer treatment and inhibitor 4 could prevent K562 cell line well from proliferation, arrest cell cycle and induce K562 towards apoptosis to achieve the goals of reversing cancer cells which could become a potential leukemia therapeutic agent in the future.

Medium Recycle Process for the Production of scu-PA from Serum Free Medium (무혈청 배지로부터 scu-PA의 생산을 위한 배지의 재사용 공정에 관한 연구)

  • 김영남;박경유
    • KSBB Journal
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    • v.8 no.1
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    • pp.17-22
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    • 1993
  • $1.96{\times}10^{-5}$(IU/cell/hr) of specific scu-PA production rate was obtained from HEK cells in maintaining ca. $8{\times}10^{5}$(cells/ml) of maximum roll density at 10(ml/hr) of perfusion rate with recycling 20% serum free conditioned media. It can be compared to $4{\times}10^{6}$(cells/ml) of maximum cell density and $4.56{\times}10^{-4}$(IU/cell/hr) of specific production rate in cultivating cells with 1% serum containing medium. Thc conversion ratio of scu-PA to tc-UK increased up to 55% as the recycling ratio increased; however, recycling the used medium seemed to have least negative effect on cell growth. It also showed that the recycling process had definitive advantage of using serum free medium in perfusion cultivation of HEK cell line.

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Purification and characterization of biochemical properties of hemolysin from Vibrio fluvialis (Vibrio fluvialis 유래의 hemolysin 정제와 생화학적 특성)

  • 이종희;한정현;안선희;김선회;이은미;공인수
    • Journal of Life Science
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    • v.12 no.4
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    • pp.490-495
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    • 2002
  • Hemolysin (VFH) of V. fluvialis, which is a pathogenic bacteria, causing watery diarrhea with vomiting, abdominal croup, was purified. V. fluvialis was cultivated in BHI medium and the culture supematant was precipitated by ammonium sulfate. The protein was purified by chromatographies on columns of DEAE-cellulose and Mono-Q. Molecular weight of the purified VFH was estimated as 79kDa by SDS-PAGE. The optimal temperature for a maximum hemolytic activity was at around 35$^{\circ}C$ and the activity was decreased at 4$0^{\circ}C$ Cytotoxicity of VFH was also investigated using RTG-2 cell line. LDH assay study showed that 50$\mu\textrm{g}$/m1 of VFH release 80% of total cellular LDH (lactate dehydrogenase) from RTG-2 cell and microscopic observation also showed the morphological change of cell.

Study on the Anti-cancer, Anti-metastasis and Immune response improvement Effects of Aqua-acupuncture with Rubi Fructus infusion solution (항암(抗癌) 및 면역효과(免疫效果)에 복분자약침(覆盆子藥鍼)이 미치는 영향(影響))

  • Lee, Seon-Hee;Lee, Byung-Ryul
    • Journal of Acupuncture Research
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    • v.17 no.1
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    • pp.189-219
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    • 2000
  • To study the effects of anti-cancer, anti-metastasis and immune response improvement effects of aqua-acupuncture with Rubi Fructus infusion solution, we used Rubi Fructus infusion solution(taken by water-alcohol method) put into Chung-wan (CV12) and Chok-Samni(ST36) of BALB/c or C57BL/6 which are corresponding to humanbody. We observed the cytotoxicity, the effect on the expression of MMP-9 gene, the ability to control cancer cell proliferation, change of body weight, surviving number, median surviving time, increase of life span, changes in amount of leukocyte, erythrocyte, platelet, total protein, creatinine, glucose and LDH, weight of spleen, number of pulmonary colony, histological analysis on tissue metastasis of lung and liver, splenic cell proliferation, the expression of cytokine gene, the number of $CD4^+$, $CD8^+$, $CD19^+$ and NK cell, and concluded like this. The results were obtained as follows : 1. Effects of Anti-cancer 1) The cytotoxicity about B16-F10 cell line of $2^0$, $2^{-1}$, $2^{-2}$, $2^{-3}$, $2^{-5}$, $2^{-6}$, $2^{-7}$, $2^{-8}$ diluent groups in Rubi Fructus infusion solution treatment was inhibited significantly, compared with control group. 2) The cytotoxicity about HT1080 cell line of $2^0{\sim}2^{-8}$ diluent groups in Rubi Fructus infusion solution treatment was inhibited significantly, compared with control group. 3) The effect on expression of MMP-9 gene was inhibited significantly in all the sample groups, compared with control group. 4) The effect on the control-ability on the cancer cell proliferation showed cytotoxicity significantly in $2^0$, $2^{-1}$, $2^{-2}$, $2^{-3}$, $2^{-4}$, $2^{-5}$, $2^{-6}$, $2^{-7}$, diluent groups. 2. Effects of Anti-metastasis 1) S-180 cancer cell line transplants in BALB/c mice were inhibited significantly in weight increase in all the sample groups, compared with control group. The surviving number increased in almost sample groups, except one group put into Chok-Samni(ST36) with 20% Rubi Fructus infusion solution treatment group that showed same number of the control group. 2) S-180 cancer cell line transplants in BALB/c mice showed high MST significantly in almost sample groups, compared with control group. But one group put into Chok-Samni(ST36) with 20% Rubi Fructus infusion solution showed low MST than control group. 3) The group injected in vein with B16-F10 cancer cell line in C57BL/6 mice showed increased ILS than control group significantly in anti-metastasis test. 3. Effects of Immune response improvement 1) The group injected in vein with B16-F10 cancer cell line in C57BL/6 mice were increased significantly in the number of leukocyte and glucose, and decreased significantly in the amount of platelet and LDH, compared with control group. However, there's no significant increase or decrease in number of erythrocyte, total protein and creatinine. 2) We couldn't find any significant relation in spleen weight of the sample group. 3) In pulmonary colony, sample group was decreased significantly, compared with control group. 4) Histological analysis of sample group inhivited compared with that of control group in both of lung and liver. 5) In immune system, all the sample groups showed having more relevancy to the effect on splenic cell proliferation than normal group. 6) Cytokine gene increased in almost sample groups, except one group treated with $50{\mu}g/m{\ell}$ Rubi Fructus infusion solution on IL-12. 7) In flow cytometry there's no significant relation in number of $CD8^+$ cell, however, the number of $CD4^+$, $CD19^+$ cell and NK cell in sample group had more relation than in control group. Above the results showed that aqua-acupuncture of Rubi Fructus solution has effects of anti-cancer, and-metastasis and immune response improvement.

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Histone Deacetylases and their Inhibitors as Potential Therapeutic Drugs for cholangiocarcinoma - Cell Line findings

  • Sriraksa, Ruethairat;Limpaiboon, Temduang
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.4
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    • pp.2503-2508
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    • 2013
  • Histone deacetylation mediated by histone deacetylases (HDACs) has been reported as one of the epigenetic mechanisms associated with tumorigenesis. The poor responsiveness of anticancer drugs found with cholangiocarcinoma (CCA) leads to short survival rate. We aimed to investigate mRNA expression of HDACs class I and II, and the effect of HDAC inhibitors, suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA), in CCA in vitro. Expression of HDACs was studied in CCA cell lines (M213, M214 and KKU-100) and an immortal cholangiocyte (MMNK1) by semi-quantitative reverse transcription-PCR. SAHA and VPA, as well as a classical chemotherapeutic drug 5 -fluorouacil (5-FU) were used in this study. Cell proliferation was determined by sulforhodamine assay. $IC_{50}$ and $IC_{20}$ were then analyzed for each agent and cell line. Moreover, synergistic potentional of VPA or SAHA in combination with 5-FU at sub toxic does ($IC_{20}$) of each agent was also evaluated. Statistic difference of HDACs expression or cell proliferation in each experimental condition was analyzed by Student's t-test. The result demonstrated that HDACs were expressed in all studied cell types. Both SAHA and VPA inhibited cell proliferation in a dose-dependent manner. Interestingly, KKU-100 which was less senstitive to classical chemotheraoeutic 5-FU was highly was sensitive to HDAC inhibitors. Simultaneous combination of subtoxic doses of HDAC inhibitors and 5-FU signiicantly inhibited cell proliferation in CCA cell lines compared to single sgent treatment($P{\leq}0.01$), while sequentially combined treatments were less effective. The present study showed inhibitory effects of HDACIs on cell proliferation in CCA cell lines, with synergistic antitumor potential demonstrated by simultaneous combination of VPA or SAHA with 5-FU, suggesting a novel alternative therapeutic strategy in effective treatment of CCA.

Effects of Substance P on the Release of Cytokines from Immune Cell Lines (면역세포의 cytokine 유리에 미치는 substance P의 영향)

  • Lee, Jin-Yong;Kim, Soo-Ah;Seo, Seok-Ran;Kim, Hyong-Seop
    • Journal of Periodontal and Implant Science
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    • v.27 no.2
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    • pp.425-441
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    • 1997
  • The neuropeptide substance P(SP) has been implicated in the mediation of inflammation and immune-mediated disease such as arthritis. Recently, it was reported that SP was markedly increased around the blood vessels in inflamed gingiva as well as in close association with the inflammatory cell infiltrate. These results support that SP may contribute to the pathophysiology of neuronal inflammation in human periodontal tissues. SP may regulate inflammatory/immune responses by stimulating the proliferation of human T cells, differentiation and antibody-secreting potential of B cells, macrophage respiratory burst, connective tissue proliferation, and the secretion of cytokines from monocytes and T cells. Here, I studied potential role of SP as a costimulatory chemical signal in inflammatory/immune responses, by determining the released proinflammatory cytokines such as $MIP-1{\alpha}$, $IL-1{\beta}$, and IL-6 from culture supernatants of homogeneous immune cell lines. Serum free cell supernatants were concentrated with TCA precipitation, fractionated with SDS-PAGE, and subjected into western blot analysis. Among 15 cell lines tested, macrophage/monocyte cell line RAW264.7 and WRl9m.1 showed the highest level of induction of $MIP-1{\alpha}$ when stimulated with LPS. Discrete IL-6 bands with multiple forms of molecular mass were detected from supernatants of B cell lines A20(32kDa), Daudi(32, 35kDa), and SKW6.4(29kDa), which were expressed constitutively. $IL-1{\beta}$ could not be detected by the method of western blot analysis from supernatants of all cell lines tested except RAW264.7, WRl9m.1, and erythroid cell line K562 which showed the least amount of $IL-{\beta}$ secretion. SP $10^{-9}M$ with suboptimal dose of LPS treatment showed synergistic induction of $MIP-1{\alpha}$ release from RAW264.7 or WR19m.1, and also IL-6 release from A20, but this synergism is not the case in costimulation of RAW264.7 or WRl9m.1 with SP $10^{-9}M$ and TPA. Although treatment of T cell line CTLL-R8 with SP $10^{-7}M$ or PHA+TPA induced modest level of $MIP-1{\alpha}$ secretion, synergism was not observed when they are applied together. These findings all together suggest the possibility of a regulatory role of SP in inflammatory/immune reaction through differential modulation of bioactivities of other chemical cosignals.

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