생명과학회지 (Journal of Life Science)

  • 제28권2호
  • /
  • Pages.176-186
  • /
  • 2018
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

인간 간암세포주 HepG2에서 김 분획물의 항산화 활성을 통한 증식 억제 및 유전자 발현 양상

Anti-proliferating Effects and Gene Expression Profiles through Antioxidant Activity of Porphyra yezoensis Fractions on Human HepG2 Cell Lines

  • 오윤정 (호서대학교 생명과학과) ;
  • 김정민 (제노플랜코리아(주) 유전체분석팀) ;
  • 방인석 (호서대학교 생명과학과)
  • Oh, Youn Jeong (Departmentof Biological Science and the Research Institute for Basic Sciences, Hoseo University) ;
  • Kim, Jung Min (Genoplan, Inc. & NAR Center, Inc.) ;
  • Bang, In Seok (Departmentof Biological Science and the Research Institute for Basic Sciences, Hoseo University)
  • 투고 : 2017.11.13
  • 심사 : 2017.12.19
  • 발행 : 2018.02.28
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

김(Porphyra yezoensis, Laver)의 MeOH 추출에 의한 유기용매 별 분획물에서 폴리페놀 함량과 항산화 활성 및 간암세포주 HepG2의 세포증식 억제효과를 확인하였다. $CHCl_3$ 분획물의 폴리페놀 함량은 $10.34{\mu}g/mg$으로 물 분획물의 $13.08{\mu}g/mg$ 보다는 다소 적게 나타났으나, DPPH 자유라디칼 소거에 의한 전자공여능(EDA)에서 나타난 $ED_{50}$은 $16.96{\mu}g/ml$로 가장 높게 나타났다. $CHCl_3$과 EtOAc 분획물은 농도의존적으로 HepG2 세포의 증식을 억제하였으며, 특히 $900{\mu}g/ml$의 $CHCl_3$ 분획물을 24시간 동안 처리하여 90%의 세포증식이 억제되었다. 한편 $CHCl_3$ 분획물이 처리된 HepG2 세포의 유전자 발현 양상을 microarray로 확인하였다. P. yezoensis의 효능과 연관지은 gene ontology 분석으로 비타민 D 합성 과정, 항균작용에 대한 반응 및 영양물질에 대한 반응에 관련된 유의 유전자들을 탐색하였다. 유의 유전자로 IL6R와 CYP1A1를 선정하였고, 이들 유전자의 상위 조절자는 ARNT 유전자가 선정되었다. 또한 50 및 $100{\mu}g/ml$의 $CHCl_3$ 분획물이 처리된 HepG2 세포에서 IL6R와 CYP1A1 단백질의 발현과 상위 조절자인 ARNT의 활성을 Western blotting으로 확인하였다.

In this study, the total polyphenol contents, antioxidant activities and anti-proliferation effects of HepG2 cell lines in organic slovent fractions obtained from the main methanolic extract of P. yezoensis were analyzed. The polyphenol content of the $CHCl_3$ fraction was $10.3{\mu}g/mg$, slightly less than $13.08{\mu}g/mg$ of the water fraction, but $ED_{50}$ estimated by measuring DPPH free radical scavenging activity exhibited the highest $16.96{\mu}g/ml$ in the $CHCl_3$ fraction. The proliferation effects of $CHCl_3$ and EtOAc fraction toward HepG2 cells inhibited in a dose-dependent manner, showed 90% inhibition when treated for 24 hr at $900{\mu}g/ml$ of $CHCl_3$ fraction. Meanwhile gene expression patterns in HepG2 cells treated $50{\mu}g/ml$ of $CHCl_3$ fraction were identified with microarray analysis. Concerning the efficacy of P. yezoensis, gene ontology analysis explored the genes associated with response to molecule of bacterial origin, vitamin D metabolic process, and response to nutrient. Thus IL6R, CYP1A1 were selected as significant genes based on expression patterns of HepG2 cells, and pathway analysis indicates that ARNT might be considered as a upstream regulator. Also, expression analysis of IL6R and CYP1A1, activity of upstream regulator ARNT in HepG2 cells was confirmed based on Western blotting analysis at the protein level after being treated with 50 and $100{\mu}g/ml$ of $CHCl_3$ fraction.

키워드

참고문헌

  1. Blois, M. S. 1958. Antioxidant determinations by the use of a stable free radical. Nature 181, 1199-1200. https://doi.org/10.1038/1811199a0
  2. Cha, M. H. and Kim, Y. K. 2008. Analysis of consumption values of a seaweed functional food. J. Kor. Soc. Food Cult. 23, 462-468.
  3. Cho, S. M., Kim, B. M., Han, K. J., Seo, H. Y., Han, Y., Yang, E. H. and Kim, D. S. 2009. Current status of the domestic processed laver market and manufacturers. Food Sci. Ind. 42, 57-70.
  4. Chopin, T., Yarish, C., Wilkes, R., Belyea, E., Lu, S. and Mathieson, A. 1999. Developing porphyra/salmon integrated aquaculture or bioremediation and diversification of the aquaculture industry. J. Appl. Phycol. 11, 463472.
  5. Cian, R. E., Martinez-Augustin, O. and Drago, S. R. 2012. Bioactive properties of peptides obtained by enzymatic hydrolysis from protein byproducts of Porphyra columbina. Food Res. Int. 49, 364-372. https://doi.org/10.1016/j.foodres.2012.07.003
  6. Hwang, E. S. and Thi, N. D. 2014. Effects of extraction and processing methods on antioxidant compound contents and radical scavenging activities of laver (Porphyra tenera). Prev. Nutr. Food Sci. 19, 40-48. https://doi.org/10.3746/pnf.2014.19.1.040
  7. Hwang, M. S., Kim, S. M., Ha, D. S., Baek, J. M., Kim, H. S. and Choi, H. G. 2005. DNA sequences and identification of Porphyra cultivated by natural seeding on the southwest coast of Korea. Algae 20, 183-196. https://doi.org/10.4490/ALGAE.2005.20.3.183
  8. Isaka, S., Cho, K., Nakazono, S., Abu, R., Ueno, M., Kim, D. and Oda, T. 2015. Antioxidant and anti-inflammatory activities of porphyran isolated from discolored nori (Porphyra yezoensis). Int. J. Biol. Macromol. 74, 68-75. https://doi.org/10.1016/j.ijbiomac.2014.11.043
  9. Ishihara, K., Oyamada, C., Matsushima, R., Murata, M. and Muraoka, T. 2005. Inhibitory effect of porphyran, prepared from dried "Nori", on contact hypersensitivity in mice. Biosci. Biotechnol. Biochem. 69, 1824-1830. https://doi.org/10.1271/bbb.69.1824
  10. Jimenez-Escrig, A. and Goni, C. I. 1999. Nutritional evaluation and physiological effects of edible seaweeds. Arch Latinoam Nutr. 49, 114-120.
  11. Jung, B. M., Shin, T. S., Kim, H. R., Jung, K. J. and Kim, S. B. 2003. Effect of porphyran isolated from laver, Porphyra yezoensis on calcium, magnesium and potassium contents in hperlipidemic rats. J. Kor. Fish. Soc. 36, 220-224.
  12. Kim, B. M., Jun, J. Y., Park, Y. B. and Jeong, I. H. 2006. Antioxidative activity of methanolic extracts from seaweeds. Kor. J. Food Nutr. 35, 1097-1101. https://doi.org/10.3746/jkfn.2006.35.8.1097
  13. Kim, D. G., Park, J. B. and Lee, T. K. 2013. Analysis of biochemical compositions and nutritive values of six species of seaweed. J. Life Sci. 23, 1004-1009. https://doi.org/10.5352/JLS.2013.23.8.1004
  14. Kim, H., Lee, Y., Han, T. and Choi, E. M. 2015. The micosporine-like amino acids-rich aqueous methanol extract of laver (Porphyra yezoensis) inhibits adipogenesis and induces apoptosis in 3T3-L1 adipocytes. Nutr. Res. Pract. 9, 592-598. https://doi.org/10.4162/nrp.2015.9.6.592
  15. Kim, S., You, D. H., Han, T. and Choi, E. M. 2014. Modulation of viability and apoptosis of UVB-exposed human keratinocyte HaCaT cells by aqueous methanol extract of laver (Porphyra yezoensis). J. Photochem. Photobiol. B, Biol. 141, 301-307. https://doi.org/10.1016/j.jphotobiol.2014.10.012
  16. Kim, Y. M., Do, J. R., In, J. P. and Park, J. H. 2005. Angiotensin Ι converting enzyme (ACE) inhibitory activities of laver (Porphyra tenera) protein hydrolysate. Kor. J. Food Nutr. 18, 11-18.
  17. Kwon, M. J. and Nam, T. J. 2006. Porphyran induces apoptosis related signal pathway in AGS gastric cancer cell lines. Life Sci. 79, 19561962.
  18. Kwon, M. J. and Nam, T. J. 2007. Chromatographically purified porphyran from Porphyra yezoensis effectively inhibits proliferation of human cancer cells. Food Sci. Biotechnol. 16, 873-878.
  19. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685. https://doi.org/10.1038/227680a0
  20. Lee, H. J., Choi, J. I. and Choi, S. J. 2012. Physiological activities and amino acid compositions of Korean dried laver porphyra products. Kor. J. Fish Aquat. Sci. 45, 409-413.
  21. Lee, J. C., Hou, M. F., Huang, H. W., Chang, F. R., Yeh, C. C., Tang, J. Y. and Chang, H. W. 2013. Marine algal natural products with anti-oxidative, anti-inflammatory, and anti-cancer properties. Cancer Cell Int. 13, 55. https://doi.org/10.1186/1475-2867-13-55
  22. Lee, M. K., Kim, I. H., Choi, Y. H. and Nam, T. J. 2015. A peptide from Porphyra yezoensis stimulates the proliferation of IEC6 cells by activating the insulinlike growth factor I receptor signaling pathway. Int. J. Mol. Med. 35, 533-538. https://doi.org/10.3892/ijmm.2014.2037
  23. Lim, S. N., Cheung, P. C. K., Ooi, V. E. C. and Ang, P. O. 2002. Evaluation of antioxidative activity of extracts from a brown seaweed, Sargassum siliquastrum. J. Agric. Food Chem. 50, 3862-3866. https://doi.org/10.1021/jf020096b
  24. Liu, F., Liu, J., Zhang, L., Shen, W., Guo, T., Liu, C. and He, P. 2008. Ex vivo antioxidation activity of polysaccharides from the red alga Porphyra yezoensis. Res. J. Pharmacol. 1, 6166.
  25. Nisizawa, K., Noda, H., Kikuchi, R. and Watanabe, T. 1987. The main seaweed foods in Japan. Hydrobiologia 151/152, 5-29. https://doi.org/10.1007/BF00046102
  26. Nunn, J. R. and von Holdt, M. M. 1957. Red-seaweed poly saccharides-2. Porphyra capensis and the separation of D-and L-galactose by crystallization. J. Chem. Soc. 215, 1094-1099.
  27. Park, S. J., Ryu, J., Kim, I. H., Choi, Y. H. and Nam, T. J. 2015. Activation of the mTOR signaling pathway in breast cancer MCF-7 cells by a peptide derived from Porphyra yezoensis. Oncol. Rep. 33, 19-24. https://doi.org/10.3892/or.2014.3557
  28. Rao, P. V. S., Mantri, V. A. and Ganesan, K. 2007. Mineral composition of edible seaweed Porphyra vietnamensis. Food Chem. 102, 215-218. https://doi.org/10.1016/j.foodchem.2006.05.009
  29. Scalbert, A., Johnson, I. T. and Saltmarsh, M. 2005. Polyphenols: antioxidants and beyond. Am. J. Clin. Nutr. 81, 215-217. https://doi.org/10.1093/ajcn/81.1.215S
  30. Shin, E. S., Hwang, H. J., Kim, I. H. and Nam, T. J. 2011. A glycoprotein from Porphyra yezoensis produces anti-inflammatory effects in liposaccharidestimulated macrophages via the TLR4 signaling pathway. Int. J. Mol. Med. 28, 809815.
  31. Shin, M. O. and Bae, S. J. 2005. Anti-proliferating effects of Porphyra tenera fractions on several cancer cell lines in vitro. J. Kor. Soc. Food Sci. Nutr. 34, 1514-1519. https://doi.org/10.3746/jkfn.2005.34.10.1514
  32. Suetsuna, K. 1998. Purification and identification of angiotensin Iconverting enzyme inhibitors from the red alga Porphyra yezoensis. J. Mar. Biotechnol. 6, 163167.
  33. Teruya, T., Konish, T., Uechi, S., Tamaki, H. and Tako, M. 2007. Anti-proliferative activity of oversulfated fucoidan from commercially cultured Cladosiphon okamuranus TOKIDA in U937 cells. Int. J. Biol. Macromol. 41, 221-226. https://doi.org/10.1016/j.ijbiomac.2007.02.010
  34. Towbin, H., Staehelin, T. and Gordon, J. 1979. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA. 76, 4350-4354. https://doi.org/10.1073/pnas.76.9.4350
  35. Vigna, L. 2015. Vitamin D supplementation promotes weight loss and waist circumference reduction in overweight/obese adults with hypovitaminosis D. European Congress on Obesity 5, 6-9.
  36. Wang, J., Zhang, Q., Zhang, Z., Song, H. and Li, P. 2010. Potential antioxidant and anticoagulant capacity of low molecular weight fucoidan fractions extracted from Laminaria japonica. Int. J. Biol. Macromol. 46, 6-12. https://doi.org/10.1016/j.ijbiomac.2009.10.015
  37. Zhang, Q., Zhang, J., Shen, J., Silva, A., Dennis, D. A. and Barrow, C. J. 2006. A simple 96-well microplate method for estimation of total polyphenol content in seaweeds. J. Appl. Phycol. 18, 445-450. https://doi.org/10.1007/s10811-006-9048-4