Journal of the Korean Applied Science and Technology (한국응용과학기술학회지)

The Korean Society of Applied Science and Technology (한국응용과학기술학회)
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Comparative Analysis on Antioxidant Activity in Various Human Skin Fibroblasts and Mesenchymal Stem Cells

사람의 피부 섬유아세포 및 중간엽 줄기세포에서 항산화 활성의 반응에 대한 비교 분석

  • Kong, Ji-Weon (Science Education Center for the Gifted, Gyeongsang National University) ;
  • Park, Ryeok (Science Education Center for the Gifted, Gyeongsang National University) ;
  • Park, Joon-Woo (Science Education Center for the Gifted, Gyeongsang National University) ;
  • Lee, Joo-Yeong (Science Education Center for the Gifted, Gyeongsang National University) ;
  • Choi, Yeon-Joo (Science Education Center for the Gifted, Gyeongsang National University) ;
  • Moon, Sun-Ha (Department of Biology Education, Gyeongsang National University) ;
  • Kim, Hyeon-Ji (Department of Biology Education, Gyeongsang National University) ;
  • Jeon, Byeong-Gyun (Science Education Center for the Gifted, Gyeongsang National University)
  • 공지원 (경상대학교 과학영재교육원) ;
  • 박력 (경상대학교 과학영재교육원) ;
  • 박준우 (경상대학교 과학영재교육원) ;
  • 이주영 (경상대학교 과학영재교육원) ;
  • 최연주 (경상대학교 과학영재교육원) ;
  • 문선하 (경상대학교 생물교육과) ;
  • 김현지 (경상대학교 생물교육과) ;
  • 전병균 (경상대학교 과학영재교육원)
  • Received : 2019.05.08
  • Accepted : 2019.06.19
  • Published : 2019.06.30
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Abstract

The cellular senescence may be due to damage by the reactive oxygen species (ROS). This study has compared the antioxidant activity in the human cell lines of various origins, including 10S and 50S-derived normal skin fibroblasts, and 10S bone marrow, dental tissue and adipose-derived adult stem cells. After being exposed to $H_2O_2$, half inhibitory concentration ($IC_{50}$) values by cytotoxicity assay was significantly (P<0.05) lower in 50S-derived skin fibroblasts, than in 10S-derived skin fibroblasts and various adult stem cell lines. The cell population doubling time (PDT) and the cell frequency with high senescence associated-${\beta}$-galactose activity were remarkably increased in 50S-derived fibroblasts exposed to 50 ppm $H_2O_2$ for 7 days, than those of 10S-derived fibroblasts and various adult stem cell lines. Further, the expression level of antioxidant-related genes, glutathione peroxidase (GPX) and catalase (CAT), was investigated in 10S and 50S-derived skin fibroblasts, and 10S-derived various adult stem cells by reverse transcription polymerase chain reaction (RT-PCR). The expression level of GPX was higher in most of cell lines, compared to CAT, and a significantly (P<0.05) higher expression level of GPX was observed in 10S-derived skin fibroblasts and adult stem cell lines, compared to 50S-derived skin fibroblasts. We concluded that old-aged skin fibroblasts seemed to be less resistant against ROS than young-aged skin fibroblasts and adult stem cells.

본 연구는 사람의 다양한 세포주를 이용하여 활성산소종(과산화수소수)이 세포의 노화에 미치는 영향을 비교 조사하였다. 여러 농도의 과산화수소수에 세포주를 일주일 동안 배양하여 MTT 방법으로 과산화수소수에 대한 세포 성장의 반억제농도를 구하였다. 그 결과, 50대에서 유래하는 피부 섬유아세포와 10대의 노화 유도 피부 섬유아세포와 비교하여 10대에서 유래하는 피부 섬유아세포에서 과산화수소수에 대한 반억제농도의 값이 유의적으로 더 높았고, 10대의 피부 섬유아세포보다는 10대의 여러 조직 기원하는 성체줄기세포에서 반억제농도의 값이 유의적으로 더 높게 관찰되었다. 또한, 50 ppm 과산화수소수를 1주일 동안 처리한 후, 50대의 피부 섬유아세포에서 다른 세포주에 비해 세포 성장이 현저히 억제되었고, 노화 관련 베타-갈락토시다아제의 활성이 증가되는 것을 관찰하였다. 또한, 활성산소의 세포 독성을 중화시키는 두 유전자, 글루타티온 과산화효소(GPX)와 카탈라아제(CAT)의 발현을 각 세포주에서 조사하였을 때, CAT의 발현은 모든 세포주에서 대체로 낮았지만, GPX 유전자의 발현이 50대의 피부 섬유아세포보다 10대의 피부 섬유아세포와 성체줄기세포에서 현저히 높게 발현되는 것을 관찰하였다. 이상의 결과에서 활성산소는 세포 노화를 유도하고, GPX의 발현이 높은 10대의 피부 섬유아세포와 줄기세포보다는 50대의 피부 섬유아세포와 노화된 피부 섬유아세포에서 활성산소종에 대해 더 큰 민감성을 가지고 있는 것을 알 수 있었다.

Keywords

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Fig. 1. MTT analysis in various human cell lines treated with H2O2.

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Fig. 2. Analysis of cell viability by MTT assay in 10S (A), 50S (B) and aged 10S (C) fibroblasts treated with H2O2.

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Fig. 3. Analysis of cell viability by MTT assay in 10S bone marrow (10S BMSC, A), third molar (10S DSC, B) and adipose (10S, ASC, C) tissue-derived mesenchymal stem cells treated with H2O2.

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Fig. 4. Determination of mean IC50 values by MTT assay in 10S fibroblasts, 50S fibroblasts, aged 10S fibroblasts, 10S BMSC, DSC and ASC treated with H2O2. a, b, c and d indicate significant (P<0.05) difference among each cell lines, respectively.

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Fig. 5. A. Analysis of population doubling time (PDT) in 10S fibroblasts, 50S fibroblasts, aged 10S fibroblasts, 10S BMSC, DSC and ASC treated with H2O2. a and b indicate significant (P<0.05) difference between untreated control (■) and treatment (■), respectively.

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Fig. 6. Morphological alterations and senescence-associated-β-galactosidase activity in 10S fibroblasts, 50S fibroblasts, aged 10S fibroblasts, 10S BMSC, DSC and ASC treated with H2O2 under inverted microscope (×200). The cells with high β-galactosidase activity were stained to blue color. Scale bars; 50 μm.

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Fig. 7. Expression level of GPX (■) and CAT (■) transcripts analyzed by RT-PCR in 10S fibroblasts, 50S fibroblasts, aged 10S fibroblasts, 10S BMSC, DSC and ASC. A, B and C indicate significant (P<0.05) difference on GPX transcript among cell lines, respectively. a, b, c and d indicate significant (P<0.05) difference on CAT transcript among cell lines, respectively.

Table 1. Primer sequence for RT-PCR

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References

  1. B. G. Childs, M. Durik, D. J. Baker, J. M. van Deursen, "Cellular senescence in aging and age-related disease: from mechanisms to therapy", Nat. Med, Vol. 21, pp. 1424-1435, (2015). https://doi.org/10.1038/nm.4000
  2. D. Fusco, G. Colloca, M. R. Lo Monaco, M. Cesari, "Effects of antioxidant supplementation on the aging process", Clin. Interv. Aging, Vol. 2, pp. 377-387, (2007).
  3. D. Pierpaola, M. Tijana, C. Andrea, L. Angela, D. Domenico, "ROS, Cell senescence, and novel molecular mechanisms in aging and age-related diseases", Oxid. Med. Cell. Longev, Vol. 2016, pp. 3565127, (2016).
  4. A. Pole, M. Dimri, G. P. Dimri, "Oxidative stress, cellular senescence and ageing", AIMS Molecular Science, Vol. 3, pp. 300-324, (2016). https://doi.org/10.3934/molsci.2016.3.300
  5. E. J. Na, H. H. Jang, G. R. Kim, "Review of recent studies and research analysis for anti-oxidant and anti-aging materials", Asian J. Beauty Cosmetol, Vol. 14, pp. 481-491, (2016). https://doi.org/10.20402/ajbc.2016.0107
  6. Q. M. Chen, "Replicative senescence and oxidant-induced premature senescence. Beyond the control of cell cycle checkpoints", Ann. N. Y. Acad. Sci, Vol. 908, pp. 111-125, (2000). https://doi.org/10.1111/j.1749-6632.2000.tb06640.x
  7. S. E. Ryu, "Do Reactive Oxygen Species Cause Aging?", Hanyang Med. Rev, Vol. 33, pp. 75-76, (2012). https://doi.org/10.7599/hmr.2013.33.2.75
  8. S. W. Kang, "Role of Reactive Oxygen Species in Cell Death Pathways", Hanyang Med. Rev, Vol. 33, pp. 77-82, (2013). https://doi.org/10.7599/hmr.2013.33.2.77
  9. H. I. Kim, S. H. Moon, W. C. Lee, H. J. Lee, S. B. Shivakumar, S. H. Lee, B. W. Park, G. J. Rho, B. G. Jeon, "Inhibition of cell growth by cellular differentiation into adipocyte-like cells in dexamethasone sensitive cancer cell lines", Anim. Cells Syst, Vol. 22, pp. 178-188, (2018). https://doi.org/10.1080/19768354.2018.1476408
  10. Y. D. Kim, S. J. Jang, E. J. Lim, J. S. Ha, S. B. Shivakumar, G. J. Jeong, G. J. Rho, B. G. Jeon, "Induction of telomere shortening and cellular apoptosis by sodium meta-arsenite in human cancer cell lines", Anim. Cell Sys, Vol. 21, pp. 241-254, (2017). https://doi.org/10.1080/19768354.2017.1342691
  11. Y. W. Kim, Y. C. Moon, W. J. Lee, S. H. Cho, G. E. Ha, B. G. Jeon, "Cytotoxic effects of sodium benzoate, food preservative in various cultured mammalian cell lines", J. Sci. Edu. Gift, Vol. 10, pp. 121-133, (2018).
  12. B. G. Jeon, D. O. Kwack, G. J. Rho, "Variation of telomerase activity and morphology in porcine mesenchymal stem cells and fibroblasts during prolonged in vitro culture", Anim. Biotechnol, Vol. 22, pp. 197-210, (2011). https://doi.org/10.1080/10495398.2011.624651
  13. E. J. Lim, H. J. Kim, M. J. Kim, S. H. Lee, B. G. Jeon, "Comparative analysis on anti-aging, anti-adipogenesis, and anti-tumor effects of green tea polyphenol epigallocatechin-3-gallate", J. Life Sci, Vol. 28, pp. 1201-1211, (2018). https://doi.org/10.5352/JLS.2018.28.10.1201
  14. F. Rodier, J. Campisi, "Four faces of cellular senescence", J. Cell Biol, Vol. 192, pp. 547-656, (2011). https://doi.org/10.1083/jcb.201009094
  15. C. M. Koch, K. Reck, K. Shao, Q. Lin, S. Joussen, P. Ziegler, G. Walenda, W. Drescher, B. Opalka, T. May, T. Brummendorf, M. Zenke, T. Saric, W. Wagner, "Pluripotent stem cells escape from senescence-associated DNA methylation changes", Genome Res, Vol. 23, pp. 248-259, (2013). https://doi.org/10.1101/gr.141945.112
  16. G. P. Dimri, X. Lee, G. Basile, M. Acosta, G. Scott, C. Roskelley, E. E. Medrano, M. Linskens, I. Rubelj, O. Pereira-Smith, "A biomarker that identifies senescent human cells in culture and in aging skin in vivo", Proc. Natl. Acad. Sci. USA, Vol. 92, pp. 9363-9367, (1995). https://doi.org/10.1073/pnas.92.20.9363
  17. J. W. Kim, S. H. Kim, J. H. Lee, "Effect of hydrogen peroxide-induced oxidative stress on the senescence of trabecular meshwork cells", J. Korean Ophthalmol. Soc, Vol. 49, pp. 1665-1670, (2008). https://doi.org/10.3341/jkos.2008.49.10.1665
  18. S. Y. Lee, H. R. Wi, M. S. Lee, "Comparison of the antioxidant effects of diallyl dulfide, capsaicin, gingerol and sulforaphane in $H_2O_2$-stressed HepG2 cells. Korean J. Nutr, Vol. 44, pp. 488-497, (2011). https://doi.org/10.4163/kjn.2011.44.6.488
  19. J. M. Mates, F. Sanchez-Jimenez, "Antioxidant enzymes and their implications in pathophysiologic processes", Front. Biosci, Vol. 4, pp. D339-345, (1995).
  20. H. J. Choi, S. H. Kim, H. T. Oh, M. J. Chung, C. B. Cui, S. S. Ham, "Effects of Adenophora triphylla ethylacetate extract on mRNA levels of antioxidant enzymes in human HepG2 cells", J. Korean Soc. Food Sci. Nutr, Vol. 37, pp. 1238-1243, (2008). https://doi.org/10.3746/jkfn.2008.37.10.1238
  21. T. W. Joo, S. H. Hong, S. Y. Park, G. Y. Kim, J. W. Jhoo JW, "Antioxidant effects of eriodictyol on hydrogen peroxide-induced oxidative stress in HepG2 cells", J. Korean Soc. Food Sci. Nutr, Vol. 45, pp. 510-517, (2016). https://doi.org/10.3746/jkfn.2016.45.4.510
  22. B. G. Jeon, S. J. Jang, J. S. Park, R. B. Subbarao, G. J. Jeong, B. W. Park, G. J. Rho, "Differentiation potential of mesenchymal stem cells isolated from human dental tissues into non-mesodermal lineage", Anim. Cell Syst, Vol. 19, pp. 321-331, (2015). https://doi.org/10.1080/19768354.2015.1087430
  23. B. G. Jeon, B. M. Kumar, E. J. Kang, S. A. Ock, S. L. Lee, D. O. Kwack, J. H. Byun, B. W. Park, G. J. Rho, "Characterization and comparison of telomere length, telomerase and reverse transcriptase activity and gene expression in human mesenchymal stem cells and cancer cells of various origins", Cell Tissue Res, Vol. 345, pp. 149-161, (2011). https://doi.org/10.1007/s00441-011-1191-9
  24. R. T. Calado, B. Dumitriu, "Telomere dynamics in mice and humans", Semin. Hematol, Vol. 50, pp. 165-174, (2013). https://doi.org/10.1053/j.seminhematol.2013.03.030
  25. Y. Zhang, M. S. Kim, B. Jia, J. Yan, J. P. Zuniga-Hertz, C. Han, D. Cai, "Hypothalamic stem cells control ageing speed partly through exosomal miRNAs", Nature, Vol. 548, pp. 52-57, (2017). https://doi.org/10.1038/nature23282
  26. E. Strozyk, D. Kulms, "The role of AKT/mTOR pathway in stress response to UV-irradiation: implication in skin carcinogenesis by regulation of apoptosis, autophagy and senescence", Int. J. Mol. Sci, Vol. 14, pp. 15260-15285, (2013). https://doi.org/10.3390/ijms140815260
  27. J. L. Kirkland, T. Tchkonia, "Cellular senescence: a translational perspective", EBio. Medicine, Vol. 21, pp. 21-28, (2017).
  28. O. M. Ighodaro, O. A. Akinloye, "First line defence antioxidants-superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX): Their fundamental role in the entire antioxidant defence grid", Alexandria Journal of Medicine, Vol. 54, pp. 287-293, (2017). https://doi.org/10.1016/j.ajme.2017.09.001
  29. A. Valle-Prieto, P. A. Conget, "Human mesenchymal stem cells efficiently manage oxidative stress", Stem Cells Dev, Vol. 19, pp. 1885-1893, (2010). https://doi.org/10.1089/scd.2010.0093
  30. L. Madhavan, V. Ourednik, J. Ourednik, "Increased "vigilance" of antioxidant mechanisms in neural stem cells potentiates their capability to resist oxidative stress", Stem Cells, Vol. 24, pp. 2110-2119, (2006). https://doi.org/10.1634/stemcells.2006-0018
  31. K. I. Kim, "Biology of aging: medical perspective", J. Korean Med. Assoc, Vol. 50, pp. 216-220, (2007). https://doi.org/10.5124/jkma.2007.50.3.216