Journal of the Korean Society of Food Science and Nutrition (한국식품영양과학회지)

The Korean Society of Food Science and Nutrition (한국식품영양과학회)
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Anticarcinogenic Responses of MCF-7 Breast Cancer Cells to Conjugated Linoleic Acid (CLA)

식이성 Conjugated Linoleic Acid (CLA)가 유선암 세포(MCF-7)에서의 항암효과에 미치는 영향

  • 문희정 (성신여자대학교 식품영양학과) ;
  • 이순재 (대구가톨릭대학교 식품영양학과) ;
  • 박수정 (성신여자대학교 식품영양학과) ;
  • 장유진 (성신여자대학교 식품영양학과) ;
  • 이명숙 (대구가톨릭대학교 식품영양학과)
  • Published : 2003.04.01
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Abstract

Conjugated linoleic acid (CLA) is the mixture of positional and geometric isomers of linoleic acid (LA, C18:2 $\omega$6), which is found abundantly in dairy products and meats. This study was peformed to investigate the anticarcinogenic effect of CLA in MCF-7 breast cancer cells. MCF-7 cell were treated with LA and CLA at the various concentrations of 15, 30, 60, 120 UM each. After incubation for 48 and 72 hours, cell proliferation, fatty acids incorporation into cell, peroxidation and activities of antioxidant enzymes were measured. Postaglandin E$_2$ (PGE$_2$) and thromboxane $A_2$ (TXA$_2$) were measured for the eicosanoids metabolism. There was no cell growth differences in both of LA and CLA treated MCF-7 cells at 48 hr incubation. Compared to LA, cell growth was decreased by CLA treatment according to increasing concentration at longer incubation times, respectively (p<0.05). Both of LA and CLA was incorporated into the cellular lipids 22~54% higher than in control but LA incorporation was not so linear as CLA according to concentration. Arachidonic acid (C20:4, $\omega$6) was synthesized after treatment of LA but did not in CLA, respectively. The lipid peroxide concentration in LA 120 $\mu$M group increased as 1.7 times as that in CLA 120 $\mu$M treated. The activities of antioxidant enzymes such as glutathione peroxidase and glutathione reductase were increased by the supplementation with CLA 120 $\mu$M at 72 hr incubation (p<0.001) compared to LA, otherwise activity of superoxide dismutase was not different in both. PGE$_2$ and TXA$_2$ levels were lower in condition of CLA treatments according to lower levels of arachidonic acids than those in LA treated group, respectively. Overall, the dietary CLA might change the MCF-7 cell growth by the changes of cell composition, production of lipid peroxide, activities of antioxidant enzymes and eicosanoid synthesis compared to dietary LA.

본 연구는 식이성 CLA 투여에 따른 MCF-7 세포에서의 항암 효과를 기전적으로 규명하여 점차 증가하는 유방암 환자의 식이 지방질의 식생활 지침서 설정에 기초 자료를 제공하는 것을 목적으로 한다. 본 연구의 결과로, 지방산 종류에 따른 증식율을 살펴보면, 48시간 배양시 LA투여군에 비하여 CLA 투여군에서 농도가 증가할수록 세포증식이 증가하는 경향이었으나 LA군과 CLA군간의 차이는 없었다. 처치 농도별로 살펴보면 LA에 비하여 72시간 배양에서 CLA농도가 15$\mu$M에서 60$\mu$M로 증가할수록 LA투여군에 비하여 증식이 감소하였고 120$\mu$M에서는 차이가 없었다. 따라서 CLA투여군은 배양시간이 길수록 농도에 따라 LA투여군보다 증식이 억제된 것으로 나타났다. 세포내 지방산 유입률은 대조군보다 LA와 CLA 투여군의 세포내 유입률이 각각 22~54%, 17~41%정도로 더 많이 유입되어 세포내 지방산 조성에 영향을 주는 것으로 보인다. LA의 경우 고농도에서 세포내로의 유입이 잘 되지 않은 것은 세포내 항상성과 관련이 있는 것으로 생각된다. 대조군보다 LA과 CLA 지방산을 48시간 배양시 지질 과산화물 생성이 50~130%증가하였고 투여량이 증가할수록 MDA의 생성이 모두 증가하였다. 그러나, 72시간 배양시 CLA 30 $\mu$M에서는 대조군과 같은 수준으로 감소하였고 120 $\mu$M에서는 LA의 과산화물 생성이 CLA군보다 1.7배가량 증가하였다. 72시간 120$\mu$M CLA처치시 항산화 효소인 GPx, GR의 활성이 높게 나타난 것은 배양시 간이 길어질수록 CLA에 대한 항산화효소 활성이 더 커지며 이는 LA군과도 큰 차이를 보였다. 따라서, CLA의 장기간 처치 경우에도 과산화물 생성이 LA군보다 적게 생성된 것은 항산화효소 활성의 역할이 있을 수 있음을 배제할 수 없겠다. 또한, 유선암 세포에 CLA를 투여 할 경우 LA투여군에 비하여 PGE$_2$와 TXA$_2$ 생성이 크게 감소하였으며 이는 AA생성의 감소와 일치하는 결과이다. 이상의 결과를 종합해 볼 때 MCF-7 유선암세포에 LA군에 비하여 CLA투여에 따른 세포증식의 차이는 없었으나 배양시간이 증가할수록 CLA농도에 따른 세포증식률이 LA군보다 감소하는 경향이 있었다. 이는 항산화효소의 증가에 의한 지질 과산화물 생성삼소와 PGE$_2$및 TXA$_2$등의 유의적인 감소가 유선암세포의 증식을 억제하는 인자로 작용할 수 있는 가능성을 시사하였다. 또한 지방산 투여 에 따른 과산화물 생성과 항산화 시스템이 유선암 세포에서는 상호기전적으로 작용하지 않았으나 이는 농도와 배양시간에 따른 CLA의 항암효과가 in vivo 실험에서 재검증 할 필요가 있음을 시사한다. 또한 장시간의 세포배양 실험시 PGE$_2$는 반감기가 짧고 다른 대사물로 쉽게 전환되는 불안전한 물질이므로 새로운 측정방법의 개발이 필요하겠다. 더욱이 CLA에 의한 항암효과가 정확히 어떤 기전에 의한 것인지 보다 깊이 있는 연구가 필요하지만 본 연구에서 얻은 에이코사노이드 대사의 변화 등은 유선암 예방 연구에 기초적인 자료가 될 것으로 생각된다.

Keywords

References

  1. Park SC, Oh SL, Lee MS. 1998. Korean status of alcoholics and alcohol related health problems. Alcohol Clin Exp Res 22: 170s-172s.
  2. Korean National Statistical Office. 2000. Annual Report on the Cause of Death Statistics.
  3. Pitot HC, Sirica AE. 1980. The stages of initiation and promotion in hepatocarcinogenesis. Biochim Biophys Acta 605: 191-215.
  4. Pariza MW, Aeschbacher HU, Felton JS, Sato S. 1990. Mutagens and carcinogens in the diet. Progress in Clinical and Biological Research 347: 217-221.
  5. Prasad K, Meyskens FL. 1990. Nutrients and cancer prevention. Human press, Clifton, NJ.
  6. Kim SH, Kim JO, Lee SH, Park KY, Chung HY. 1991. Antimutagenic compounds identified from the chloroform fraction of garlic (Allium sativum). J Korean Soc Food Nutr 20: 253-259.
  7. Ha YL, Pariza MW. 1990. Anticarcinogenic conjugated derivatives of linoleic acid found in grilled ground beef: isolation, identification, and mechanism of action. In First Korean Conference on Science and Technology. Korean Fed Sci Assoc, Seoul, Korea. p 442.
  8. Ha YL, Storkson JM, Pariza MW. 1990. Inhibition of benzos [a]pyrene-induced mouse forestomach neoplasia by conjugated dienoic derivatives of linoleic acid. Cancer Res 50: 1097-1011.
  9. Pariza MW, Ha YL. 1990. Conjugated dienoic derivatives of linoleic acid: A new class of anticarcinogens. Med Oncol Tumor Phamacother 7: 169-171.
  10. Pariza MW, Ha YL. 1990. Conjugated dienoic derivatives of linoleic acid: Mechanism of anticarcinogenic effect. In Mutagens and carcinogens in the diet. Pariza MW, Aeschbacher HU, Felton JS, Sato S, eds. WilleyLiss, Inc., New York. p 217.
  11. Park HS. 2000. Mechanism of cancer prevention and other physiological function by CLA. Korean J Nutrition 33: 556-565.
  12. Ip C, Shin M, Thompson HJ, Scimeca JA. 1994. Conjugated linoleic acid suppresses mammary carcinogenesis and proliferative activity of the mammary gland in the rat. Cancer Res 54: 1212-1215.
  13. Ip C, Chin SF, Scimeca JA, Pariza MW. 1991. Mammary cancer prevention by conjugated dienoic derivative of linoleic acid. Cancer Res 51: 6118-6124.
  14. Visonneau S, Cesano A, Tepper SA, Scimeca JUA, Santoli D, Kritchevsky D. 1997. Conjugated linoleic acid suppresses the growth of human breast adenocarcinoma cells in SCID mice. Anticancer Res 17: 969-974. https://doi.org/10.1097/01.cad.0000224446.31577.df
  15. Ip C, Carter CA, Ip MM. 1985. Requirement of essential fatty acid for mammary tumorigenesis. Cancer Res 45: 1997-2000.
  16. Diane CC, Lisa YH, Terry DS. 1997. Proliferative responses of normal human mammary and MCF-7 breast cancer cells to linoleic acid, conjugated linoleic acid and eicosanoid synthesis inhibitors in culture. Anticancer Res 17: 197-204.
  17. Shultz TD, Chew BP, Seaman WR. 1992. Differential stimulatory and inhibitory response of human MCF-7 breast cancer cells to linoleic acid and conjugated linoleic acid in culture. Anticancer Res 12: 2143-2146.
  18. Ip C, Juang C, Thompson HJ, Scimeca JA. 1997. Retention of conjugated linoleic acid in the mammary gland is associated with tumor inhibition during the post-initiation phase on carcinogenesis. Carcinogenesis 18: 755-759. https://doi.org/10.1093/carcin/18.4.755
  19. Vijayender RD, Gabriel F. 1997. The growth inhibitory effect of conjugated linoleic acid on MCF-7 cells is released to estrogen response system. Cancer Lett 116: 121-130. https://doi.org/10.1016/0304-3835(82)90052-0
  20. Van den Berg JJM, Cook NE, Tribble DL. 1995. Reinvestigation of the antioxidant properties of conjugated linoleic acid. Lipid 30: 599-605. https://doi.org/10.1007/BF02536996
  21. Banni S, Angioni E, Contini MS, Carta G, Casu V, Iengo GA, Melis MP, Diana M, Dessi MA, Corongiu FP. 1998. Conjugated linoleic acid and oxidative stress. J Am Oil Chem Soc 75: 261-267. https://doi.org/10.1007/s11746-998-0040-9
  22. Lee MS. 2000. Apolipoprotein metabolism. Hyoil Co., Seoul. p 265-295.
  23. Alfthan G, Aro A, Arvilommi H, Huttunen JK. 1991. Selenium metabolism and platelet glutathione peroxidase activity in healthy Finnish men: effects of selenium yeast, selenite and selenate. Am J Clin Nutr 53: 120-125. https://doi.org/10.1093/ajcn/53.1.120
  24. Vane JR, Mitchell JA, Appleton I, Tomlinson A, Bailey DB, Croxtall J, Willoughby DA. 1994. Inducible isoforms of cyclooxygenase and nitric oxide synthase in inflammation. Proc Natl Acad Sci USA 91: 2046-2050. https://doi.org/10.1073/pnas.91.6.2046
  25. Kai-Li L, Martha AB. 1998. Conjugated linoleic acid reduces arachidonic acid content and PGE2 synthesis in murine keratinocytes. Cancer Letters 127: 15-22. https://doi.org/10.1016/S0304-3835(97)00479-5
  26. Scudiero DA, Shoemaker RH, Paull KD, Monks A, Tierney S, Nofziger TH, Currens MJ, Seniff D, Boyd MR. 1988. Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines. Cancer Res 48: 4827-33.
  27. Van Grembrock MMJ, Voorhout WF, Erkelen DW, van Meer G, de Braun TAW. 1995. Palmitic acid and linoleic acid metabolism in Caco-2 cells; different triglyceride synthesis and lipoprotein secretion. J Lipid Res 36: 13-24.
  28. Folch J, Lees M, Shoane-Stanley GH. 1957. A simple method for the isolatioon and purification of total lipids from animal tissue. J Biol Chem 226: 497-509.
  29. Packer L, Smith J. 1974. Extension of the lifespan of cultured human diploid cells by vitamin E. Proc Natl Acad Sci USA 71: 4763-4767. https://doi.org/10.1073/pnas.71.12.4763
  30. Cavanagh EMV, Inserra F, Ferder L, Romano L, Ercole L, Fraga CG. 1996. Superoxide dismutase and glutathione per-oxidase activities are increased by enalapril and captopril in mouse liver. FEBS Lett 361: 21-24.
  31. Carlberg I, Mennervick B. 1985. Glutathione reductase. Meth Enzymol 113: 484-450.
  32. McCord JM, Fridovich I. 1969. Superoxide dismutase-An enzymatic function for erythrocuperin (hemocuperin). J Biol Chem 224: 6049-6055.
  33. Kim WK. 1997. Efffect of different sources of dietary n-3 fatty acids on immune response and eicosanoids production. J Korean Soc Fooc Sci Nutr 26: 697-703.
  34. Yang JA, Kim SO, Choi JH, Kwag OG, Rhee SJ, Chang HW. 1998. Activities of phospholipase $A_2$ and cyclooxygenase, and syntheses of thromboxane and prostacyclin in streptozotocin induced diabetic rats. J Korean Soc Fooc Sci Nutr 27: 175-181.
  35. Bradford MM. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-54. https://doi.org/10.1016/0003-2697(76)90527-3
  36. Rose DP, Connolly JM. 1989. Stimulation of growth of human breast cancer cell lines in culture by linoleic acid. Biochem Biophys Res Commun 164: 277-283. https://doi.org/10.1016/0006-291X(89)91714-2
  37. Martha A, Anna KS. 1997. Conjugated linoleic acid modulates hepatic lipid composition in mice. Lipids 32: 199-204. https://doi.org/10.1007/s11745-997-0025-0
  38. Cook ME, Miller CC, Park Y, Pariza M. 1993. Immune modulation by altered nutrient metabolism: Nutritional control of immune-induced growth depression. Poultry Sci 72: 1301-1305. https://doi.org/10.3382/ps.0721301
  39. John JT, Young L, Ingrid AS, Ken GDA, Bruce AW. 1998. Modulation of macrophage cytokine production by conjugated linoleic acids is influenced by the dietary n-6 : n-3 fatty acid ratio. J Nutr Biochem 9: 258-266. https://doi.org/10.1016/S0955-2863(98)00014-X
  40. Ip C, Stephanie PB, Alvert DH, Henry JT, Jayne S, Joseph AS. 1996. The efficiency of conjugated linoleic acid in mammary cancer prevention is independent of the level or type of fat in the diet. Carcinogenesis 17: 1045-1050. https://doi.org/10.1093/carcin/17.5.1045
  41. Hartley JA, Gibson NW, Qwelling LA, Yuspa SH. 1985. The association of DNA strand breaks with accelerated terminal differentiation in mouse epidermal cells exposed to tumor promoters. Cancer Res 45: 4864-4871.
  42. Song HJ, Park HS. 1999. Effect of conjugate linoleic acid on hepatic peroxidation and antioxidant enzyme system in 1,2-dimethylhydrazine-treated rats. MS Thesis. Kyunghee University.
  43. Kwon SY, Lee MS. 2000. The effect of feeding conjugated linoleic acid (CLA) on antioxidant enzymes system in S.D. rats. MS Thesis. Sungshin Women's University.
  44. Helen C, Rosaleen D, Catherine S, Feragal L. 1998. The effect of a conjugated linoleic acid on superoxide dismutase, catalase and glutathione peroxidase in oxidatively-challenged liver cells. Biochemical Society Transactions S62: 26.
  45. Marianne O, Catherine S, Rosaleen D. 1999. Antioxidant enzyme defence responses of human MCF-7 and SW480 cancer cell to conjugated linoleic acid. Cancer Res 19: 1953-1960.
  46. Sebedio JL, Juaneda P, Dobson G, Ramilison I, Martin JC, Chardigny JM, Christie WW. 1997. Metabolites of conjugated isomers of linoleic acid (CLA) in the rat. Biochim Biophys Acta 1345: 5-10.
  47. Claudine JK, Kai-Li L, Martha AB. 1999. Effect of dietary conjugated linoleic acid on phorbol ester-induced $PGE_2$ production and hyperplasia in mouse epidermis. Nutrition and Cancer 33: 132-138. https://doi.org/10.1207/S15327914NC330203
  48. Anne T, Gerald M, Jack YV. 1999. Antiplatelet effects of conjugated linoleic acid isomers. Biochim Biophys Acta 1438: 239-246 https://doi.org/10.1016/S1388-1981(99)00055-4
  49. Abou-EI-Ela SH, Prasse KW, Carroll R, Wade AE, Dharwadkar S, Bounce OR. 1988. Eicosanoid synthesis in 7, 12-dimethylbenz(a)anthracene-induced mammary adenocarcinomas in Sprague-Dawley rats fed primrose oil, menhaden oil or corn oil diet. Lipid 23: 948-954. https://doi.org/10.1007/BF02536342