Utilization of IκB–EGFP Chimeric Gene as an Indicator to Identify Microbial Metabolites with NF-κB Inhibitor Activity

Biological Procedures Online

Yu-Ling Lin¹  Yen-Shun Chen⁴  Jui-Hung Hsieh²  Ching-Min Lin¹  Hsin-Yi Wu¹  Chen-Si Lin⁶  Rea-Min Chu⁶  Kuang-Wen Liao³  Yuan-Hsun Hsu⁵ 

[1] Institute of Molecular Medicine and Bioengineering, National Chiao Tung University, Hsinchu, Taiwan

[2] Department of Electronics Engineering and Institute of Electronics, National Chiao Tung University, Hsinchu, Taiwan

[3] Department of Biological Science and Technology, National Chiao Tung University, 300 Room 205 Zhu-Ming Building, 75 Bo-Ai Street, Hsinchu, Taiwan

[4] Graduate Institute of Medical Sciences, Taipei Medical University, Taipei, Taiwan

[5] Department of Microbiology and Immunology, College of Medicine, Taipei Medical University, Taipei, Taiwan

[6] Animal Cancer Center, School of Veterinary Medicine, National Taiwan University, Taipei, Taiwan

关键词: Propolis;    Hydroquinone;    EGFP;    IκB;    Antioxidant;    Microbial metabolite;   

Others  :  793413
DOI  :  10.1007/s12575-010-9033-9

 received in 2010-04-03, accepted in 2010-09-15,  发布年份 2010

【 摘 要 】

NF-κB regulates several important expressions, such as cytokine release, anti-apoptosis, adhesion molecule expression, and cell cycle processing. Several NF-κB inhibitors have been discovered as an anti-tumor or anti-inflammatory drug. The activity of NF-κB transcription factor is negatively regulated by IκB binding. In this study, IκB assay system was established and IκB–EGFP fusion protein was used as an indicator to monitor the effects of substances on the IκB degradation. The results indicated that the chosen hydroquinone could inhibit the IκB degradation and cause the cell de-attachment from the bottom of culture plate. In addition, this system could also monitor the IκB degradation of microbial metabolite of natural mixtures of propolis. Thus, the IκB assay system may be a good system for drug discovery related to microbial metabolite.

【 授权许可】

   
2010 Lin et al; licensee Springer

【 图 表 】

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【 参考文献 】

• [1]Pahl HL: Activators and target genes of Rel/NF-kappaB transcription factors. Oncogene 1999, 18:6853-6866.
• [2]Ghosh S, May MJ, Kopp EB: NF-kappa B and Rel proteins: evolutionarily conserved mediators of immune responses. Annu Rev Immunol 1998, 16:225-260.
• [3]Bowie A, O'Neill LA: Oxidative stress and nuclear factor-kappaB activation: a reassessment of the evidence in the light of recent discoveries. Biochem Pharmacol 2000, 59:13-23.
• [4]Ho E, Bray TM: Antioxidants, NFkappaB activation, and diabetogenesis. Proc Soc Exp Biol Med 1999, 222:205-213.
• [5]Janssen YM, Sen CK: Nuclear factor kappa B activity in response to oxidants and antioxidants. Meth Enzymol 1999, 300:363-374.
• [6]Weber C, Erl W: Modulation of vascular cell activation, function, and apoptosis: role of antioxidants and nuclear factor-kappa B. Curr Top Cell Regul 2000, 36:217-235.
• [7]Christman JW, Blackwell TS, Juurlink BH: Redox regulation of nuclear factor kappa B: therapeutic potential for attenuating inflammatory responses. Brain Pathol 2000, 10:153-162.
• [8]Henkel T, Machleidt T, Alkalay I, Kronke M, Ben-Neriah Y, Baeuerle PA: Rapid proteolysis of IκB-alpha is necessary for activation of transcription factor NF-κB. Nature 1993, 365:182-185.
• [9]Li X, Fang Y, Zhao X, Jiang X, Duong T, Kain ST: Characterization of NF-κB activation by detection of green fluorescent protein-tagged IkB degradation in living cells. J Biol Chem 1999, 274:21244-21250.
• [10]Lernbecher T, Müller U, Wirth T: Distinct NF-kappa B/Rel transcription factors are responsible for tissue-specific and inducible gene activation. Nature 1993, 365:767-770.
• [11]Ahmad M, Marui N, Alexander RW, Medford RM: Cell type-specific transactivation of the VCAM-1 promoter through an NF-kappa B enhancer motif. J Biol Chem 1995, 270:8976-8983.
• [12]Phelps CB, Sengchanthalangsy LL, Huxford T, Ghosh G: Mechanism of I kappa B alpha binding to NF-kappa B dimers. J Biol Chem 2000, 275:29840-29846.
• [13]Heissmeyer V, Krappmann D, Wulczyn FG, Scheidereit C: NF-kappaB p105 is a target of IkappaB kinases and controls signal induction of Bcl-3–p50 complexes. EMBO J 1999, 18:4766-4778.
• [14]Ma Q, Kinneer K, Ye J, Chen BJ: Inhibition of nuclear factor kappaB by phenolic antioxidants: interplay between antioxidant signaling and inflammatory cytokine expression. Mol Pharmacol 2003, 64:211-219.
• [15]Kim E, Kang BY, Kim TS: Inhibition of interleukin-12 production in mouse macrophages by hydroquinone, a reactive metabolite of benzene, via suppression of nuclear factor-kappaB binding activity. Immunol Lett 2005, 99:24-29.
• [16]Ouyang Y, Virasch N, Hao P, Aubrey MT, Mukerjee N, Bierer BE, Freed BM: Suppression of human IL-1beta, IL-2, IFN-gamma, and TNF-alpha production by cigarette smoke extracts. J Allergy Clin Immunol 2000, 106:280-287.
• [17]Pyatt DW, Yang Y, Stillman WS, Cano LL, Irons RD: Hydroquinone inhibits PMA-induced activation of NFkappaB in primary human CD19+ B lymphocytes. Cell Biol Toxicol 2000, 16:45-51.
• [18]Karin M, Cao Y, Greten FR, Li ZW: NF-kappaB in cancer: from innocent bystander to major culprit. Nat Rev Cancer 2002, 2:301-310.
• [19]Wang Y, Chen Q, Zeng X: Potentiometric biosensor for studying hydroquinone cytotoxicity in vitro. Biosens Bioelectron 2010, 25:1356-1362.
• [20]Cho JY: Suppressive effect of hydroquinone, a benzene metabolite, on in vitro inflammatory responses mediated by macrophages, monocytes, and lymphocytes. Mediat Inflamm 2008, 2008:1-11.
• [21]Lin MW, Yang SR, Huang MH, Wu SN: Stimulatory actions of caffeic acid phenethyl ester, a known inhibitor of NF-kappaB activation, on Ca2+-activated K+ current in pituitary GH3 cells. J Biol Chem 2004, 279:26885-26892.