Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Antioxidative and Anticancer Activities of Julbernardia globiflora Extract in Human Colon Adenocarcinoma HT29 Cells

Julbernardia globiflora 추출물의 항산화 활성 및 인체 대장암 세포 HT29에 대한 항암 활성 분석

  • Oh, You Na (Blue-Bio Industry Regional Innovation Center, Dong-Eui University) ;
  • Jin, Soojung (Blue-Bio Industry Regional Innovation Center, Dong-Eui University) ;
  • Kwon, Hyun Ju (Blue-Bio Industry Regional Innovation Center, Dong-Eui University) ;
  • Kim, Byung Woo (Blue-Bio Industry Regional Innovation Center, Dong-Eui University)
  • 오유나 (동의대학교 블루바이오소재개발센터) ;
  • 진수정 (동의대학교 블루바이오소재개발센터) ;
  • 권현주 (동의대학교 블루바이오소재개발센터) ;
  • 김병우 (동의대학교 블루바이오소재개발센터)
  • Received : 2016.12.12
  • Accepted : 2017.02.02
  • Published : 2017.05.30
Download PDF
⟨ Previous Next ⟩

Abstract

Julbernardia globiflora, a tropical African tree widespread in Miombo woodland, has been used in folk medicine for the treatment of depression and stomach problems. However, the antioxidative and anticancer activities of J. globiflora remain unclear. The objective of this study is to evaluate the antioxidative and anticancer effects of methanol extract of J. globiflora (MEJG) and the molecular mechanism of its anticancer activity in human colon carcinoma HT29 cells. MEJG exhibited significant antioxidative effect with an $IC_{50}$ (concentration at 50% inhibition) value of $1.23{\mu}g/ml$ measuring by 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay and inhibited cell proliferation in a dose-dependent manner in HT29 cells. We found that MEJG induced apoptosis of HT29 cells with the increase of apoptotic cells and apoptotic bodies using Annexin V staining and 4,6-diamidino-2-phenylindole (DAPI) staining, respectively. The MEJG treatment showed the increase of Fas, a death receptor, and Bax, a pro-apoptotic protein, and the decrease of Bcl-2, an anti-apoptotic protein, resulting in the release of cytochrome c from the mitochondria into the cytosol and activation of caspase-3, -8 and -9. The apoptotic effects of MEJG were confirmed by cleavage of poly (ADP-ribose) polymerase (PARP). Collectively, these results suggest that MEJG may exert the anticancer effect in HT29 cells by inducing apoptosis via both the intrinsic and extrinsic pathways.

Julbernardia globiflora는 미옴보 숲에 널리 분포하는 열대 아프리카 나무로, 우울증, 위장장애 등의 치료를 위해 민간요법으로 사용되고 있으나, 항산화능, 항암 활성 등에 대한 연구는 알려진 바가 없다. 따라서 본 연구에서는 J. globiflora 메탄올 추출물(MEJG)을 사용하여 항산화능 및 인체 대장암 세포주인 HT29에 대한 항암 활성에 관하여 분석하였다. 먼저 DPPH radical scavenging activity를 통해 분석한 결과, MEJG의 $IC_{50}$는 $1.23{\mu}g/ml$로 강력한 항산화능을 보유하였음을 확인하였다. 또한 MEJG 농도 의존적으로 HT29 세포의 성장을 억제하였다. MEJG의 HT29 세포 사멸 효과의 기전을 분석하기 위하여 Annexin V 염색과 DAPI 염색을 수행한 결과, 대조군에 비하여 apoptotic 세포 및 apoptotic body가 증가됨을 확인하였다. 또한 apoptosis 관련 단백질들의 발현변화를 분석한 결과, MEJG처리에 의해 사멸수용체인 Fas와 pro-apoptotic 단백질인 Bax의 발현이 증가되었으며, anti-apoptotic 단백질인 Bcl-2의 발현이 감소하였다. 이러한 결과로 cytochrome c가 미토콘드리아에서 세포질로 방출되어 증가되었으며, caspase-3, -8, -9가 활성화되었다. 최종적으로 PARP가 분해되어 apoptosis가 유도되었음을 확인하였다. 이러한 결과들로부터 MEJG는 내인성 및 외인성 경로를 통한 apoptosis 유도에 의하여 HT29 세포의 증식을 억제하는 항암활성을 보유하였음을 확인하였다.

Keywords

References

  1. Adam, J. M. and Cory, S. 2007. The Bcl-2 apoptotic switch in cancer development and therapy. Oncogene 26, 1324-1337. https://doi.org/10.1038/sj.onc.1210220
  2. Bhanot, A., Sharma, R. and Noolvi, M. N. 2011. Natural sources as potential anti-cancer agents: A review. Int. J. Phytomedicine 3, 9-26.
  3. Carson, D. A. and Ribeiro, J. M. 1993. Apoptosis and disease. Lancet 341, 1251-1254. https://doi.org/10.1016/0140-6736(93)91154-E
  4. Choi, I. P. 2013. Reactive oxygen species and cancer. Hanyang Med. Rev. 33, 118-122. https://doi.org/10.7599/hmr.2013.33.2.118
  5. Devasagayam, T. P., Tilak, J. C., Boloor, K. K., Sane, K. S., Ghaskadbi, S. S. and Lele, R. D. 2004. Free rardicals and antioxidants in human health: current status and future prospects. J. Assoc. Physicians India 52, 794-804.
  6. Dickinson, B. C. and Chang, C. J. 2011. Chemistry and biology of reactive oxygen species in signaling or stress responses. Nat. Chem. Biol. 7, 504-511. https://doi.org/10.1038/nchembio.607
  7. Elmore, S. 2007. Apoptosis: a review of programmed cell death. Toxicol. Pathol. 35, 495-516. https://doi.org/10.1080/01926230701320337
  8. Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L. and Hensen, P. M. 1992. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207-2216.
  9. Fluda, S. 2015. Targeting apoptosis for anticancer therapy. Semin. Cancer Biol. 31, 84-88. https://doi.org/10.1016/j.semcancer.2014.05.002
  10. Fluda, S. and Debatin, K. M. 2006. Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy. Oncogene 25, 4798-4811. https://doi.org/10.1038/sj.onc.1209608
  11. Galluzzi, L., Lopez-Soto, A., Kumar, S. and Kroemer, G. 2016. Caspases connect cell-death signaling to organismal homeostasis. Immunity 44, 221-231. https://doi.org/10.1016/j.immuni.2016.01.020
  12. Goodsell, D. S. 2000. The molecular perspective: caspases. Oncologist 5, 435-436. https://doi.org/10.1634/theoncologist.5-5-435
  13. Halliwell, B. H. and Gutteridge, J. M. C. 1990. Role of free radicals and catalytic metal ions in human disease: an overview. Methods Enzymol. 186, 1-85.
  14. Han, S. I., Kim, Y. S. and Kim, T. H. 2008. Role of apoptotic and necrotic cell death under physiologic conditions. BMB Rep. 41, 1-10. https://doi.org/10.5483/BMBRep.2008.41.1.001
  15. Hengartner, M. O. 2000. The biochemistry of apoptosis. Nature 407, 770-776. https://doi.org/10.1038/35037710
  16. Kaufmann, S. H., Desnoyers, S., Ottaviano, Y., Davidson, N. E. and Poirier, G. G. 1993. Specific proteolytic cleavage of poly (ADP-ribose) polymerase: an early marker of chemotherapy- induced apoptosis. Cancer Res. 53, 3976-3985.
  17. Koopman, G., Reutelingsperger, C. P., Kuijten, G. A., Keehnen, R. J., Pals, S. T. and van Oers, M. H. 1994. Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84, 1415-1420.
  18. Landis-Piwowar, K. R. and Iyer, N. R. 2014. Cancer chemoprevention: current state of the art. Cancer Growth Metastasis 7, 19-25.
  19. Li, P., Nijhawan, D. and Wang, X. 2004. Mitochondrial activation of apoptosis. Cell 116, S57-59. https://doi.org/10.1016/S0092-8674(04)00031-5
  20. Lowe, S. W. and Lin, A. W. 2000. Apoptosis in cancer. Carcinogenesis 21, 485-495. https://doi.org/10.1093/carcin/21.3.485
  21. Martin, S. J., Reutelingsperger, C. P., McGahon, A. J., Rader, J. A., van Schie, R. C., LaFace, D. M. and Green, D. R. 1995. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of bcl-2 and abl. J. Exp. Med. 182, 1545-1556. https://doi.org/10.1084/jem.182.5.1545
  22. Neergheen, V. S., Bahorun, T., Taylor, E. W., Jen, L. S. and Aruoma, O. I. 2010. Targeting specific cell signaling transduction pathways by dietary and medicinal phytochemicals in cancer chemoprevention. Toxicology 278, 229-241. https://doi.org/10.1016/j.tox.2009.10.010
  23. Oh, C. M., Won, Y. J., Jung, K. W., Kong, H. J., Cho, H., Lee, J. K., Lee, D. H. and Lee, K. H. 2016. Cancer statistics in Korea: Incidence, mortality, survival, and prevalence in 2013. Cancer Res. Treat. 48, 436-450. https://doi.org/10.4143/crt.2016.089
  24. Oh, Y. N., Jin, S., Park, H. J., Kim, B. Y. and Kwon, H. J. 2015. Anti-oxidative and anti-cancer activities of Treculia Africana extract in human colon adenocarcinoma HT29 cells. J. Life Sci. 25, 515-522. https://doi.org/10.5352/JLS.2015.25.5.515
  25. Palgrave, K. C., Drummond, R. B., Moll, E. J. and Palgrave, M. C. 1997. Trees of Southern Africa. Struik Publishers: Cape Town, South Africa.
  26. Porter, A. G. and Janicke, R. U. 1999. Emerging roles of caspase- 3 in apoptosis. Cell Death Differ. 6, 99-104. https://doi.org/10.1038/sj.cdd.4400476
  27. Riedl, S. J. and Shi, Y. 2004. Molecular mechanisms of caspase regulation during apoptosis. Nat. Rev. Mol. Cell Biol. 5, 897-907.
  28. Shi, L., Nishioka, W. K., Th'ng, J., Bradbury, E. M., Litchfield, D. W. and Greenberg, A. H. 1994. Premature p34cdc2 activation required for apoptosis. Science 263, 1143-1145. https://doi.org/10.1126/science.8108732
  29. Shi, Y. 2004. Caspase activation: revisiting the induced proximity model. Cell 117, 855-858. https://doi.org/10.1016/j.cell.2004.06.007
  30. Singh, S., Singh, P. P., Roberts, L. R. and Sanchez, W. 2014. Chemopreventive strategies in hepatocellular carcinoma. Nat. Rev. Gastroenterol. Hepatol. 11, 45-54. https://doi.org/10.1038/nrgastro.2013.143
  31. Stewart, B. W. and Wild, C. P. 2014. World cancer report 2014. International Agency for Research on Cancer (IARC), Geneva, World Health Organization.
  32. Strasser, A. and Newton, K. 1999. FADD/MORT1, a signal transducer that can promote cell death or cell growth. Int. J. Biochem. Cell Biol. 31, 533-537. https://doi.org/10.1016/S1357-2725(99)00003-5
  33. Taylor, R. C., Cullen, S. P. and Martin, S. J. 2008. Apoptosis: controlled demolition at the cellular level. Nat. Rev. Mol. Cell Biol. 9, 231-241. https://doi.org/10.1038/nrm2312
  34. Tsujimoto, Y. and Shimizu, S. 2000. Bcl-2 family: life-ordeath switch. FEBS Lett. 466, 6-10. https://doi.org/10.1016/S0014-5793(99)01761-5
  35. Watt, J. M. and Breyer-Brandwijk, M. G. 1962. The medicinal and poisonous plants of southern and eastern Africa. pp. 1457, 2nd ed. E. and S. Livingstone: London, United Kingdom.
  36. Yin, S. Y., Wei, W. C., Jian, F. Y. and Yang, N. S. 2013. Therapeutic applications of herbal medicines for cancer patients. Evid. Based Complement Alternat. Med. 2013, 302426.
  37. Zou, H., Li, Y., Liu, X. and Wang, X. 1999. An APAF-1 cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J. Biol. Chem. 274, 11549-11556. https://doi.org/10.1074/jbc.274.17.11549