Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Construction and In vitro Study of a Prx 6/Luc Vector System for Screening Antioxidant Compounds in the Transgenic Mice

항산화반응을 유발하는 물질의 검색에 적용할 수 있는 형질전환 마우스 생산을 위한 새로운 Prx 6/Luc 벡터시스템의 제조 및 폐암세포주에서 반응성 확인

  • Lee, Young Ju (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Nam, So Hee (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Kim, Ji Eun (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Hwang, In Sik (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Lee, Hye Ryun (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Choi, Sun Il (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Kwak, Moon Hwa (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Lee, Jae Ho (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Jung, Young Jin (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • An, Beum Soo (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University) ;
  • Hwang, Dae Youn (Department of Biomaterials Science, College of Natural Resources & Life Science, Pusan National University)
  • 이영주 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 남소희 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 김지은 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 황인식 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 이혜련 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 최선일 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 곽문화 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 이재호 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 정영진 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 안범수 (부산대학교 생명자원과학대학 바이오소재과학과) ;
  • 황대연 (부산대학교 생명자원과학대학 바이오소재과학과)
  • Received : 2012.06.01
  • Accepted : 2013.01.16
  • Published : 2013.02.28
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Abstract

Peroxiredoxin 6 (Prx 6) is a member of the thiol-specific antioxidant protein family, which may play a role in protection against oxidative stress and in regulating phospholipid turnover. The aim of this study was to determine whether a human Prx 6/Luc vector was stably expressed and responded to antioxidants in a lung cell line (NCI-H460). To achieve this, the luciferase signal, hPrx 6 mRNA expression, and superoxide dismutase (SOD) activity were measured in transfectants with a hPrx 6/Luc plasmid after treatment with four antioxidant extracts, including Korea white ginseng (KWG), Korea red ginseng (KRG), Liriope platyphylla (LP), and red Liriope platyphylla (RLP). First, the hPrx 6/Luc plasmid was successfully constructed with DNA fragments of human Prx 6 promoter, amplified by PCR using genomic DNA isolated from NCI-H460 cells, and cloned into the pTransLucent reporter vector. The orientation and sequencing of the hPrx 6/Luc plasmid were identified with restriction enzyme and automatic sequencing. A luciferase assay revealed significant enhancement of luciferase activity in the four treatment groups compared with a vehicle-treated group, although the ratio of the increase was different within each group. The KRG- and LP-treated groups showed higher activity than the KWG- and RLP-treated groups. Furthermore, the luciferase activity against RLP occurred roughly in a dose-dependent manner. However, the level of endogenous hPrx 6 mRNA did not change in any group treated with the four extracts. The SOD activity was in agreement with the luciferase activity. Therefore, these results indicate that the hPrx 6/Luc vector system may successfully express and respond to antioxidant compounds in NCI-H460 cells. The data also suggest that the Prx 6/Luc vector system may be effectively applied in screening the response of hPrx 6 to antioxidant compounds in transgenic mice.

Peroxiredoxin 6 (Prx 6)는 티올-특이적 항산화 단백질에 속하는 효소로서 산화적 스트레스로부터 세포를 보호할 뿐만 아니라 과산화물의 환원작용을 촉매한다. 본 연구에서는 인간 Prx 6의 promoter를 이용하여 항산화반응을 유발하는 추출물을 효과적으로 스크리닝하는 새로운 형질전환마우스를 개발하는 최종목적을 달성하기 위한 중간단계로서, hPrx 6/Luc 벡터를 개발하고, 이들 벡터의 안정적 발현과 성공적 반응성을 세포주를 이용하여 확인하고자 하였다. 이를 위해, 인간 Prx 6 promoter를 증폭하여 luciferase cDNA와 결합한 hPrx 6/Luc 벡터를 제조하였으며, 제조된 벡터를 제한효소 절단과 염기서열분석을 통해 확인하였다. hPrx 6/Luc 벡터는 NCI-H460 세포에 transfection한 후 인삼(KWG), 홍삼(KRG), 맥문동(LP), 홍문동(RLP)의 4가지 추출물을 처리하여 luciferase activity를 측정하였다. 그 결과, luciferase activity는 4가지 추출물에 의해 효과적으로 증가하였고, 특히 KRG과 LP를 처리한 그룹이 KWG과 RLP를 처리한 그룹보다 높았다. 또한, luciferase activity는 RLP 농도에 의존적으로 증가하였다. hPrx 6/Luc 벡터와 hPrx 6 mRNA반응의 차이를 비교하기 위해, 4가지 추출물을 처리한 후 hPrx 6 mRNA의 양을 RT-PCR로 분석하였다. 그러나, hPrx 6 mRNA의 양은 비록 고농도의 RLP 추출물에서는 약간의 증가가 관찰되었지만, 대조군에 비하여 4가지 추출물에서 유의적인 차이가 없었다. 한편, 4가지 추출물에 의한 superoxide dismutase (SOD) 활성의 변화는 비록 일부 차이는 있었지만 hPrx 6/Luc 벡터와 유사한 반응을 나타내었다. 따라서, 이러한 결과는 hPrx 6/Luc 벡터는 성공적으로 제조되었고, 세포내에서 안정적으로 발현하면서 항상화물질에 민감하고 정량적으로 반응할 수 있음을 제시하고 있다. 더불어, 이러한 세포주에서 확인결과를 바탕으로 형질전환마우스가 개발된다면, 항산화물질을 정량적으로 스크리닝하는 시스템으로 적용가능성이 매우 높음을 보여주고 있다.

Keywords

References

  1. Bae, K. M., Park, S. H., Jung, K. H., Kim, M. J., Hong, S. H., Song, Y. O. and Lee, H. S. 2010. Effects of roasting conditions on physicochemical properties and sensory properties of Liriopis Tuber. J Korean Soc Food Sci Nutr 39, 1503-1508. https://doi.org/10.3746/jkfn.2010.39.10.1503
  2. Borgstahl, G. E., Parge, H. E., Hickey, M. J., Johnson, M. J., Boissinot, M., Hallewell, R. A., Lepock, J. R., Cabelli, D. E. and Tainer, J. A. 1996. Human mitochondrial manganese superoxide dismutase polymorphic variant Ile58Thr reduces activity by destabilizing the tetrameric interface. Biochemistry 35, 4287-4297. https://doi.org/10.1021/bi951892w
  3. Chae, H. Z., Kang, S. W. and Rhee, S. G. 1999. Isoforms of mammalian peroxiredoxin that reduce peroxides in presence of thioredoxin. Methods Enzymol 300, 219-226. https://doi.org/10.1016/S0076-6879(99)00128-7
  4. Chen, X. C., Zhou, Y. C., Chen, Y., Zhu, Y. G., Fang, F. and Chen, L. M. 2005. Ginsenoside Rg1 reduces MPTP-induced substantia nigra neuron loss by suppressing oxidative stress. Acta Pharmacol Sin 26, 56-62. https://doi.org/10.1111/j.1745-7254.2005.00019.x
  5. Choi, S. I., Lee, H. R., Goo, J. S., Kim, J. E., Nam, S. H., Hwang, I. S., Lee, Y. J., Prak, S. H., Lee, H. S., Lee, J. S., Jang, I. S., Son, H. J. and Hwang, D. Y. 2011. Effects of steaming time and frequency for manufactured red Liriope platyphylla on the insulin secretion ability and insulin receptor signaling pathway. Lab Anim Res 27, 117-126 https://doi.org/10.5625/lar.2011.27.2.117
  6. Deng, H. L. and Zhang, J. T. 1991. Anti-lipid peroxilative effect of ginsenoside Rb1 and Rg1. Chin Med J (Engl) 104, 395-398.
  7. Fisher, A. B., Dodia, C., Manevich, Y., Chen, J. W. and Feinstein, S. I. 1999. Phospholipid hydroperoxides are substrates for non-selenium glutathione peroxidase. J Biol Chem 274, 21326-21334. https://doi.org/10.1074/jbc.274.30.21326
  8. Gallagher, B. M. and Phelan, S. A. 2007. Investigating transcriptional regulation of Prdx6 in mouse liver cells. Free Radic Biol Med 42, 1270-1277. https://doi.org/10.1016/j.freeradbiomed.2007.01.023
  9. Halliwell, B. 1999. Free Radicals in Biology and Medicine. 3rd. Eds., Oxford University Press. New York.
  10. Henkle-Dhrsen, K. and Kampkctter, A. 2001. Antioxidant enzyme families in parasitic nematodes. Mol Biochem Parasitol 114, 129-142. https://doi.org/10.1016/S0166-6851(01)00252-3
  11. Hess, A., Wijayanti, N., Neuschafer-Rube, A. P., Katz, N., Kietzmann, T. and Immenschuh, S. 2003. Phorbol ester-dependent activation of peroxiredoxin I gene expression via a protein kinase C, Ras, p38 mitogen-activated protein kinase signaling pathway. J Biol Chem 278, 45419-45434. https://doi.org/10.1074/jbc.M307871200
  12. Hong, C. E. and Lyu, S. Y. 2011. Anti-inflammatory and anti-oxidative effects of Korean red ginseng extract in human keratinocytes. Immune Netw 11, 42-49. https://doi.org/10.4110/in.2011.11.1.42
  13. Jiang, T., Huang, B. K., Zhang, Q. Y., Han, T., Zheng, H. C. and Qin, L. P. 2007. Effect of Liriope platyphylla total saponin on learning, memory and metabolites in aging mice induced by D-galactose. Zhong Xi Yi Jie He Xue Bao 5, 670-674. https://doi.org/10.3736/jcim20070614
  14. Kang, S. W., Bainess, I. C. and Rhee, S. G. 1998. Characterization of mammalian peroxiredoxin that contain one conserved cysteine. J Biol Chem 273, 6303-6311. https://doi.org/10.1074/jbc.273.11.6303
  15. Kang, S. W., Chae, H. Z., Seo, M. S., Kim, K., Bainess, I. C. and Rhee, S. G. 1998. Mammalian peroxiredoxin isoforms can reduce hydrogen peroxide generated in response to growth factors and tumor necrosis factor-alpha. J Biol Chem 273, 6297-6302. https://doi.org/10.1074/jbc.273.11.6297
  16. Kim, H. S., Manevich, Y., Feinstein, S. I., Pak, J. H., Ho, Y. S. and Fisher, A. B. 2003. Induction of 1-cys peroxiredoxin expression by oxidative stress in lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 285, L363-L369.
  17. Kubo, E., Fatma, N., Akagi, Y., Beier, D. R., Singh, S. P. and Singh, D. P. 2008. TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity. Am J Physiol Cell Physiol 294, C842-C855. https://doi.org/10.1152/ajpcell.00540.2007
  18. Ku¨min, A., Huber, C., Ru¨licke, T., Wolf, E. and Werner, S. 2006. Peroxiredoxin 6 is a potent cytoprotective enzyme in the epidermis. Am J Pathol 169, 1194-1205. https://doi.org/10.2353/ajpath.2006.060119
  19. Lee, S. K., Park, J. H. and Kim, Y. T. 2009. A Study on the antioxidation and antimicrobial effect of "Megmoondong (Liriope platyphylla Wang et Tang)" water extracts. J Korean Soc Food Sci Nutr 22, 279-285.
  20. Liao, B., Newmark, H. and Zhou, R. 2002. Neuroprotective effects of ginseng total saponin and ginsenosides Rb1 and Rg1 on spinal cord neurons in vitro. Exp Neurol 173, 224-234. https://doi.org/10.1006/exnr.2001.7841
  21. Liu, M. and Zhang, J. T. 1995. Protective effects of ginsenoside Rb1 and Rg1 on cultured hippocampal neurons. Yao Xue Xue Bao 30, 674-678.
  22. McGonigle, S., Dalton, J. P. and James, E. R. 1998. Peroxidoxins: a new antioxidant family. Parasitol Today 14, 139-145. https://doi.org/10.1016/S0169-4758(97)01211-8
  23. Mo, Y., Feinstein, S. I., Manevich, Y., Zhang, Q., Lu, L., Ho, Y. S. and Fisher, A. B. 2003. 1-Cys peroxiredoxin knock-out mice express mRNA but not protein for a highly related intronless gene. FEBS Lett 555, 192-198. https://doi.org/10.1016/S0014-5793(03)01199-2
  24. Nagy, N., Malik, G., Fisher, A. B. and Das, D. K. 2006. Targeted disruption of peroxiredoxin 6 gene renders the heart vulnerable to ischemia-reperfusion injury. Am J Physiol Heart Circ Physiol 291, H2636-H2640. https://doi.org/10.1152/ajpheart.00399.2006
  25. Okado-Matsumoto, A., Matsumoto, A., Fujii, J. and Taniguchi, N. 2000. Peroxiredoxin VI is a secretale protein with heparin binding properties under reduced conditions. J Biochem 127, 493-501. https://doi.org/10.1093/oxfordjournals.jbchem.a022632
  26. Radad, K., Gille, G., Moldzio, R., Saito, H. and Rausch, W. D. 2004. Ginsenosides Rb1 and Rg1 effects on mesencephalic dopaminergic cells stressed with glutamate. Brain Res 1021, 41-53. https://doi.org/10.1016/j.brainres.2004.06.030
  27. Rahman, I., Li X. Y., Donaldson, K., Harrison, D. J. and MacNee. W. 1995. Glutathione homeostasis in alveolar epithelial cells in vitro and lung in vivo under oxidative stress. Am J Physiol 269(3 Pt 1), L285-L292.
  28. Robinson, M. W., Hutchinson, A. T., Dalton, J. P. and Donnelly, S. 2010. Peroxiredoxin: a central player in immune modulation. Parasite Immunol 32, 305-313. https://doi.org/10.1111/j.1365-3024.2010.01201.x
  29. Seo, M. K., Kang, S. W., Kim, K., Baines, I. C., Lee, T. H. and Rhee, S. G. 2000. Identification of a new type of mammalian peroxiredoxin that forms an intramolecular disulfide as a reaction intermediate. J Biol Chem 275, 20346-20354. https://doi.org/10.1074/jbc.M001943200
  30. Vaca-Paniagua, F., Parra-Unda, R. and Landa, A. 2009. Characterization of one typical 2-Cys peroxiredoxin gene of taenia solium and taenia crassiceps. Parasitol Res 105, 781-787 https://doi.org/10.1007/s00436-009-1461-6
  31. Wang, X., Phelan, S. A., Forsman-Semb, K., Taylor, E. F., Petros, C., Brown, A., Lerner, C. P. and Paigen, B. 2003. Mice with targeted mutation of peroxiredoxin 6 develop normally but are susceptible to oxidative stress. J Biol Chem 278, 25179-25190. https://doi.org/10.1074/jbc.M302706200
  32. Wang, Y., Feinstein, S. I., Manevich, Y., Ho, Y. S. and Fisher, A. B. 2004. Lung injury and mortality with hyperoxia are increased in peroxiredoxin 6 gene-targeted mice. Free Radic Biol Med 37, 1736-1743. https://doi.org/10.1016/j.freeradbiomed.2004.09.006
  33. Wang, Y., Feinstein, S. I., Manevich, Y., Ho, Y. S. and Fisher, A. B. 2006. Peroxiredoxin 6 gene-targeted mice show increased lung injury with paraquat-induced oxidative stress. Antioxid Redox Signal 8, 229-237. https://doi.org/10.1089/ars.2006.8.229
  34. Wood, Z. A., Sch0072cder, E., Robin Harris, J. and Poole, L. B. 2003. Structure, mechanism and regulation of peroxiredoxins. Trends Biochem Sci 28, 32-40. https://doi.org/10.1016/S0968-0004(02)00003-8
  35. Choi, C. Y., Kim, J. Y., Kim, Y. S., Chung, Y. C., Seo, J. K., Jeong, H. G. 2001. Aqueous extract isolated from Platycodon grandiflorum elicits the release of nitric oxide and tumor necrosis factor-alpha from murine macrophages. Int Immunopharmacol 1, 1141-1151. https://doi.org/10.1016/S1567-5769(01)00047-9
  36. Kijima, I., Phung, S., Hur, G., Kwok, S. L., Chen, S. 2006. Grape seed extract is an aromatase inhibitor and a suppressor of aromatase expression. Cancer Res 66, 5960-5967. https://doi.org/10.1158/0008-5472.CAN-06-0053
  37. Xu, K., Wang, X., Tian, C., Shi, S., Wang, G. R., Shi, Q., Li, P., Zhou, R. M., Jiang, H. Y., Chu, Y. L., Dong, X. P. 2010. Transient expressions of doppel and its structural analog prionDelta32-121 in SH-SY5Y cells caused cytotoxicity possibly by triggering similar apoptosis pathway. Mol Biol Rep 37, 2549-2558. https://doi.org/10.1007/s11033-009-9772-3
  38. Yang, Y., Ikezoe, T., Nishioka, C., Bandobashi, K., Takeuchi, T., Adachi, Y., Kobayashi, M., Takeuchi, S., Koeffler, H. P., Taguchi, H. 2006. NFV, an HIV-1 protease inhibitor, induces growth arrest, reduced Akt signalling, apoptosis and docetaxel sensitisation in NSCLC cell lines. Br J Cancer 95, 1653-1662. https://doi.org/10.1038/sj.bjc.6603435