Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
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Establishment of an In Vitro TCD (Testosterone Compound Detection) System

테스토스테론 물질 검출을 위한 in vitro TCD 시스템 구축

  • Lee, Dong-Geun (Major in Pharmaceutical Engineering, Division of Bioindustry, Silla University) ;
  • Jo, Jung-Kwon (Department of Bioscience, Graduate School, Silla University) ;
  • Lee, Sang-Hyeon (Major in Pharmaceutical Engineering, Division of Bioindustry, Silla University)
  • 이동근 (신라대학교 제약공학과) ;
  • 조정권 (신라대학교 일반대학원 생명과학과) ;
  • 이상현 (신라대학교 제약공학과)
  • Received : 2019.07.10
  • Accepted : 2019.08.02
  • Published : 2019.10.30
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Abstract

Although there is a growing interest in male menopause, a phenomenon associated with male hormone depletion, current kits using antibodies to quantify male hormones are expensive. In this study, we constructed an in vitro system for verifying the activity or concentration of male steroid hormones using a transcriptional activity test. A reporter plasmid, pGL2-Neo-ARE-AdE1BTATA, which reacts to testosterone, was constructed. In this plasmid, the ARE-AdE1bTATA sequences can be bounded by the testosterone - androgen receptor complex to express luciferase as a reporter. Then, a stable transfection was performed on the human prostate cancer cell line, LNcap-LN3. The constructed LNcap-LN3/pGL2-Neo-ARE-AdE1BTATA testosterone compound detection (TCD) system showed quantitatively proportional luciferase activities to concentrations of $10^{-13}$ to $10^{-8}M$ of standard testosterone. The established in vitro TCD system will contribute to the development of materials for health/functional foods and drugs as it will be possible to search en masse for testosterone-like or testosterone-inhibiting substances derived from natural materials.

남성호르몬 감소와 관련된 남성갱년기에 대한 관심이 고조되고 있지만, 남성호르몬의 정량을 위해 항체를 이용하는 고가의 kit가 이용되고 있다. 본 연구에서는 in vitro 전사 활성 시험법을 이용하여 남성 스테로이드호르몬의 활성 혹은 농도를 검증하는 시스템을 구축하였다. 테스토스테론-AR (androgen receptor) 복합체와 반응하는 ARE-AdE1bTATA 염기서열이 삽입되고 리포터로 luciferase를 발현하는 테스토스테론 유사활성 검증 리포터 플라스미드인 pGL2-Neo-ARE-AdE1BTATA를 제조하고, 인체 전립선암 세포인 LNcap-LN3 세포에 stable transfection을 실시하였다. 구축된 LNcap-LN3/pGL2-Neo-ARE-AdE1BTATA TCD (testosterone compound detection) 시스템은 표준물질인 테스토스테론의 $10^{-13}{\sim}10^{-8}M$ 범위에서 농도 증가에 비례하는 정량성을 보였다. 이 연구에서 확립된 in vitro TCD 시스템을 이용하면 천연물 유래 테스토스테론 유사물질 및 테스토스테론 저하물질의 대량 탐색 등이 가능할 것이므로, 건강기능성 식품이나 의약품 신소재의 개발에 기여할 것이다.

Keywords

References

  1. Allgood, V. E., Oakley, R. H. and Cidlowski, J. A. 1993. Modulation by vitamin B6 of glucocorticoid receptor-mediated gene expression requires transcription factors in addition to the glucocorticoid receptor. J. Biol. Chem. 268, 20870-20876. https://doi.org/10.1016/S0021-9258(19)36867-X
  2. Handa, R. J., Roselli, C. E. and Resko, J. A. 1998. Distribution of androgen receptor in microdissected brain areas of the female baboon (Papio cynocephalus). Brain Res. 29, 111-116. https://doi.org/10.1016/0006-8993(71)90421-5
  3. http://www.index.go.kr/potal/info/idxKoreaView.do?idx_cd=2758
  4. http://www.nifds.go.kr/brd/m_15/view.do?seq=10043
  5. https://www.khsa.or.kr/user/info/InfoHealthFood.do?_menuNo=225
  6. Jeon, M. J., Kim, S., Kim, B. K., Cheon, J., Park, S. H., Oh, E., Lee, S. H. and Kim, M. 2013. The effects of seaweed gongjindan on estrogen like activities, platelet aggregation and serum lipid levels in ovariectomized rats. J. Life Sci. 23, 1155-1162. https://doi.org/10.5352/JLS.2013.23.9.1155
  7. Kim, E. T., Lee, J. H., Jung, J. H., Jo, H. W., Ju, H. T., Park, J. S., Woo, S. H. and Kim, D. K. 2010. Testosterone replacement in men with late-onset hypogonadism symptoms and loss of diurnal rhythm of total testosterone. Kor. J. Androl. 28, 190-196.
  8. Kim, G. Y., Lee, H. G. and Kim, E. J. 2015. Effects of extracts from oja on testosterone synthesis in Leydig cells. J. Physiol. Pathol. Kor. Med. 29, 403-408. https://doi.org/10.15188/kjopp.2015.10.29.5.403
  9. Kim, J. J. 2004. PADAM (Partial Androgen Deficiency in the Aging Male) and hormone replacement therapy. Kor. J. Anndrol. 22, 45-51
  10. Kim, K. M. 2013. Late-onset hypogonadism. Kor. J. Fam. Pract. 3, 245-254
  11. Kim, M. H., Kim, B. K., Kim, J. D., Kang, A. R., Lee, C. E., Seo, J. M. , Lee, D. G., Jo, J. K., Kim, Y. Y., Yu, K. H. and Lee, S. H. 2017. The effects of medicinal herbs extracts on estrogen-like activities and osteoblast proliferation and differentiation. J. Life Sci. 27, 456-463. https://doi.org/10.5352/JLS.2017.27.4.456
  12. Kim, T. M., Jung, H. K., Jang, J. H., Sim, M. O., Lee, M. J., Cho, J. H. and Cho, H. W. 2016. Effect of Corni fructus on testosterone deficiency syndrome in in vitro and in vivo. Kor. J. Pharmacogn. 47, 264-272.
  13. Laczka, O., del Campo, F. J., Munoz-Pascual, F. X. and Baldrich, E. 2011. Electrochemical detection of testosterone by use of three-dimensional disc-ring microelectrode sensing platforms: application to doping monitoring. Anal. Chem. 83, 4037-4044. https://doi.org/10.1021/ac1031594
  14. Lee, K. S., Lee, E. K., Kim, S. Y., Kim, T. H. and Kim, H. P. 2015. Effect of a mixed extract of fenugreek seeds and Lespedza cuneata on testosterone deficiency syndrome. Kor. J. Food Sci. Technol. 47, 492-498. https://doi.org/10.9721/KJFST.2015.47.4.492
  15. Marhefka, C. A., Moore, B. M., Bishop, T. C., Kirkovsky, L., Mukherjee, A., Dalton, J. T. and Miller, D. D. 2001. Homology modeling using multiple molecular dynamics simulations and docking studies of the human androgen receptor ligand binding domain bound to testosterone and nonsteroidal ligands. J. Med. Chem. 44, 1729-1740. https://doi.org/10.1021/jm0005353
  16. Min, K. S. and Kang, D. L. 2011. Testosterone replacement therapy and monitoring for the male patients with testosterone deficiency syndrome. J. Kor. Med. Assoc. 54, 197-204. https://doi.org/10.5124/jkma.2011.54.2.197
  17. Park, J. S., You, G. D., Seo, S. M., Han, S. B., Hong, J. T. and Han, K. 2013. Inhibition effect of testosterone metabolism of some natural products containing yacon and their ameliorative effect of benign prostatic hyperplasia symptom. Yakhak Hoeji 57, 241-249.
  18. Park, S. H., Jeon, M. J., Jang, M. K., Lee, S. J., Kim, B. K., Jeon, M. J., Kim, S. Y., Kim, M. H., Lee, D. G., Lee, T. H., Nam, J. S., Hur, W. J. and Lee, S. H. 2013. Verification of estrogen like activties of herbal medicines using an in vitro detection system. Kor. J. Ori. Physiol. Pathol. 27, 752-758.
  19. Seo, K. K., Yoon, S. Y. and Kim, S. C. 1998. Efficacy and safety of AndrodermTM in partially androgen-deficiency aging men with erectile dysfunction. Kor. J. Androl. 16, 79-85.