Journal of the East Asian Society of Dietary Life (동아시아식생활학회지)

The East Asian Society of Dietary Life (동아시아식생활학회)
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Effects of Ixeris dentata Extract on Radical Oxygen Species and Bcl-2 Family in Human Breast Cancer Cells

씀바귀 추출물이 인체유방암세포의 활성 산소 및 Bcl-2 Family에 미치는 영향

  • Kim, Hee-Jung (Dept. of Food and Nutrition, Duksung Women's University) ;
  • Kang, Keum-Jee (Dept. of Food and Nutrition, Duksung Women's University)
  • 김희정 (덕성여자대학교 자연과학대학 식품영양학과) ;
  • 강금지 (덕성여자대학교 자연과학대학 식품영양학과)
  • Received : 2014.09.11
  • Accepted : 2014.12.17
  • Published : 2014.12.31
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Abstract

The aim of the study was to determine the effects of Ixeris dentata extract (IDE) on anticancer activity in human breast cancer MDA-MB-231 cells at both cellular and molecular levels. The cells were cultured in the presence of 0, 20, 30 and $40{\mu}g/mL$ Ixeris dentata extract for 24 hours, respectively. At the end of culture, cytochemical analyses for MTT activity, trypan blue dye exclusion, Annexin V-FITC Apoptosis, and radical oxygen species (ROS) were conducted. RT-PCR was also performed to determine whether or not alterations in cell viability affect the Bax/Bcl-2 ratio. MTT assay showed that relative cell viability decreased in a dose-dependent manner (p<0.05). Reduction of cell viability matched well with increased cell membrane permeability as determined by trypan blue dye exclusion test (p<0.05). The rates of intracellular ROS also increased in a similar manner to those of TB-stained cells. There was an associated shift of apoptotic cells from early to late apoptosis between the 30 and $40{\mu}g/mL$. Bax/Bcl-2 ratio significantly increased along with significant decreases in Bcl-2 expression between 30 and $40{\mu}g/mL$ groups (p<0.05). In conclusion, anticancer activity of Ixeris dentata extract is modulated by a reduction in cell viability along with increased membrane permeability, leading to ROS accumulation within cells, and subsequently cell death through an apoptotic pathway that involves Bax and Bcl-2 in human breast cancer MDA-MB-231 cells.

본 연구는 인체유방암세포인 MDA-MB-231세포를 이용하여 IDE를 처리하였을 때 세포사멸에 미치는 영향을 세포화학적 방법으로 확인하였다. IDE를 각각 0, 20, 30 및 $40{\mu}g/mL$ 첨가하여 24시간 배양한 후, 세포증식 억제, 막투과성, 세포내 ROS 분석 및 세포사멸 단계의 특성을 FACS 분석하고, RT-PCR에 의한 사멸관련 유전자 중 Bax/Bcl-2 ratio 분석을 통하여 IDE의 항암작용의 조절작용을 밝히고자 하였다. MTT의 세포 증식 억제는 첨가된 IDE의 농도에 따라 유의적으로 감소하였다(p<0.05). 이와 동시에, trypan blue에 대한 염색성과 DCF-DA 형광 분석의 결과는 각각, 막투과성과 세포내 ROS 농도가 모두 농도 의존적으로 증가됨을 보였다(p<0.05). 이와 같은 IDE 농도에 따른 세포화학적 변화 중에서 세포의 초기사멸에서 후기사멸 과정으로 급격한 사멸단계의 변화가 특히, IDE 농도 30과 $40{\mu}g/mL$에서 일어났다. RT-PCR 분석에 의한 Bax/Bcl-2 ratio도 IDE 농도 30과 $40{\mu}g/mL$에서 급격히 증가하였다(p<0.05). 이와 같은 세포화학적 결과와 RT-PCR 결과를 종합해 볼 때, IDE의 유방암세포(MDA-MB-231)에 대한 세포사멸작용은 막 투과성의 증가와 ROS 증가를 통하여 세포에 점진적인 손상을 일으키며, 이는 세포의 생화학적 변화도 초래하여 세포 증식 억제를 감소시켜, 결국 세포내의 사멸관련 유전자 지표인 Bax/Bcl-2 ratio를 크게 변화시키는 일련의 세포사멸을 점진적으로 유도시켜 항유방암의 효과의 가능성을 제시하였다.

Keywords

References

  1. Abhyankar G, Suprasanna P, Pandey BN, Mishra KP, Rao KV, Reddy VD (2010) Hairy root extract of Phyllanthus amarus induces apoptotic cell death in human breast cancer cells. Innov Food Sci Emerging Technol 11: 526-532. https://doi.org/10.1016/j.ifset.2010.02.005
  2. Ahn EM, Bang MH, Song MC, Park MH, Kim HY, Kwon BM, Baek NI (2006) Cytotoxic and ACAT-inhibitory sesquiterpene lactones from the root of Ixeris dentata forma albiflora. Arch Pharm Res 29: 937-941. https://doi.org/10.1007/BF02969274
  3. Alnermri ES (1997) Mammalian cell death proteases: A family of highly conserved aspartate specific cysteine proteases. J Cell Biochem 64: 32-42.
  4. American Institute of Cancer Research/World Cancer Research Fund (2007) Food, nutrition, physical activity, and prevention of cancer: A global perspective. American Institute of Cancer Research, Washington, DC, USA. pp 41-45.
  5. Cabiscol E1, Piulats E, Echave P, Herrero E, Ros J (2000) Oxidative stress promotes specific protein damage in Saccharomyces cerevisiae. J Biol Chem 275: 27393-27398.
  6. Cha MR, Choi YH, Choi CW, Yoo DS, Kim YS, Choi SU, Kim YH, Ryu SY (2011) New guaiane sesquiterpene lactones from Ixeris dentata. Planta Med 77: 380-382. https://doi.org/10.1055/s-0030-1250369
  7. Chung HS, Jeong HJ, Han MJ, Park ST, Seong KK, Baek SH, Jeong DM, Kim MJ, Kim HM (2002) Nitric oxide and tumor necrosis factor-alpha production by Ixeris dentata in mouse peritoneal macrophages. J Ethnopharmacol 82: 217-222. https://doi.org/10.1016/S0378-8741(02)00188-5
  8. Chung HS, Woo WS, Lim SJ (1994) Dentatins: Sesquiterpene glucosides from Ixeris dentata. Arch Pharm Res 17: 323-326. https://doi.org/10.1007/BF02974170
  9. Heo BK, Chon SU, Park YJ, Bae JH, Park SM, Park YS, Jang HG, Gorinstein S (2009) Antiproliferative activity of Korean wild vegetables on different human tumor cell lines. Plant Foods Hum Nutr 64: 257-263. https://doi.org/10.1007/s11130-009-0138-8
  10. Hong S, Jeong D, Kim K, Hwang E (2011) The composition of the root of Ixeris dentata var. albiflora Nakai. and cell viability and DPPH radical scavenging activities of its extract. Korean J Nutr 43: 105-113.
  11. Kelsey JL (2005) Breast cancer epidemiology. Cancer Res 48: 5615-5623.
  12. Kim JK, Woo HJ, Kim JH, Yoon YD (2004) Effects of Ixeris dentata extracts on the genotoxicity induced by gamma irradiation in rats. Korean J Environ Biol 22: 543-549.
  13. Kim MJ, Kim JS, Jeong DM, Ham SS, Yu CY (2002a) Effect of antioxidant, antimutagenecity and anticancer of root extract from Ixeris dentata Nakai. Korean J Medicinal Crop Sci 10: 222-229.
  14. Kim MJ, Kim JS, Kang WH, Jeong DM (2002b) Effect on antimutagenic and cancer cell growth inhibition of Ixeris dentata Nakai. Korean J Medicinal Crop Sci 10: 139-143.
  15. Kim SH (1995) Inhibitory effects of Ixeris dentata on mutagenecity of aflatoxin B1, N-methyl-N'-nitro-N-nitrosoguanidine and the growth of MG-63 human osteosarcoma cells. J Korean Soc Food Nutr 24: 305-312.
  16. Kma L (2013) Roles of plant extracts and constituents in cervical cancer therapy. Asian Pacific J Cancer Prev 14: 3429-3436. https://doi.org/10.7314/APJCP.2013.14.6.3429
  17. Lee E (2011) Effects of Ixeris dentata extract on the production of pro-inflammatory cytokines in the LPS stimulated rat and Raw 264.7 cells. Korean J Plan Res 24: 604-612. https://doi.org/10.7732/kjpr.2011.24.5.604
  18. Lee KS, Kim GH, Kim HH, Kim ES, Park HM, Oh MJ (2009) Physiological functionalities of tea thermally processed from Ixeris dentata root. J Korean Soc Food Sci Nutr 38: 496-501. https://doi.org/10.3746/jkfn.2009.38.4.496
  19. Lee SN, Kang KJ (2008) The effect of blueberry on ROS accumulation and cell death in human normal breast epithelial (MCF10A) and breast cancer (MCF7) cells. Korean J Food Nutr 21: 416-424.
  20. Lim SS, Lee JH (1997) Effects of Aster scaber and Ixeris dentata on contractility and vasodilation of cardiovascular and endothelial cell in hyperlipidemic rat. J Korean Soc Food Sci Nutr 26: 300-307.
  21. Liu Y, Peterson DA, Kimura H, Schubert D (1997) Mechanism of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction. J Neurochem 69: 581-593.
  22. Ministry of Health and Walfare (2012) National survey of cancer patients: Analysis of national cancer survey. Ministry of Health and Welfare, Seoul. Republic of Korea. pp 8-15.
  23. Nogueira V1, Hay N (2013) Molecular pathways: reactive oxygen species homeostasis in cancer cells and implications for cancer therapy. Clin Cancer Res 19: 4309-4314. https://doi.org/10.1158/1078-0432.CCR-12-1424
  24. Oltvai ZN, Milliman CL, Korsmeyer SJ (1993) Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax that accelerates programmed cell death. Cell 74: 609-619. https://doi.org/10.1016/0092-8674(93)90509-O
  25. Reed JC (1994) Bcl-2 and the regulation of programmed cell death. J Cell Biol 124: 1-6. https://doi.org/10.1083/jcb.124.1.1
  26. Somasagara RR, Hegde M, Chiruvella KK, Musini A, Choudhary B, Raghavan SC (2012) Extracts of strawberry fruits induce intrinsic pathway of apoptosis in breast cancer cells and inhibits tumor progression in mice. PLoS ONE 7: e47021. https://doi.org/10.1371/journal.pone.0047021
  27. Strober W (2001) Trypan blue exclusion test of cell viability. Curr Protoc Immunol A.3B.1-A.3B.2.
  28. Trachootham D, Lu W, Ogasawara MA, Valle NRD, Huang P (2008) Redox regulation of cell survival. Antioxid Redox Signal 10: 1343-1374. https://doi.org/10.1089/ars.2007.1957
  29. Uttara B, Singh AV, Zamboni P, Mahajan RT (2006) Oxidative stress and neurodegenerative diseases: A review of upstream and downstream antioxidant therapeutic options. Curr Neuropharmacol 7: 65-74.
  30. Vaux DL (1993) Toward an understanding of the molecular mechanism of physiological cell death. Proc Natl Acad Sci 90: 786-789. https://doi.org/10.1073/pnas.90.3.786
  31. Wang J, Yi J (2008) Cancer cell killing via ROS: To increase or decrease, that is the question. Cancer Biol Thera 7: 1875-1884. https://doi.org/10.4161/cbt.7.12.7067
  32. Yang J, Liu X, Bhalla K, Kim CN, Ibrado AM, Cai J (1997) Prevention of apoptosis by bcl-2: release of cytochrome c from mitochondria blocked. Science 275: 1129-1132. https://doi.org/10.1126/science.275.5303.1129
  33. Zhang YC, He CN, Chew EH (2002) Studies on the chemical constituents and biological activities of Ixeris. Chem Biodivers 10: 1373-1391.
  34. Zhap ZQ, Budde JM, Morris C, Wang NP, Velez DA, Murak S, Guyton RA, Vinten JJ (2001) Adenosine attenuated reperfusion induced apoptotic cell death by modulation expression of Bcl-2 and Bax proteins. J Mol Cell Cardiol 33: 57-68. https://doi.org/10.1006/jmcc.2000.1275

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