Breast Cancer Research and Treatment

, Volume 55, Issue 2, pp 147–158

Synergistic action of apoptosis induced by eicosapentaenoic acid and TNP‐470 on human breast cancer cells

  • Daigo Yamamoto
  • Yasuhiko Kiyozuka
  • Yasushi Adachi
  • Hideho Takada
  • Koshiro Hioki
  • Airo Tsubura


The effects of eicosapentaenoic acid (EPA) and an angiogenesis inhibitor (TNP‐470) on the suppression of breast cancer cell growth were examined in five human breast cancer cell lines (MDA‐MB‐231, T‐47D, MCF‐7, KPL‐1, and MKL‐F). In all five cell lines, EPA and TNP‐470 alone both showed tumor growth inhibition in a time‐ and dose‐dependent manner, and in combination, a synergistic effect was seen at high concentrations. EPA plus TNP‐470 treatment evoked apoptosis as confirmed by the appearance of sub G1 populations, by DNA fragmentation, and by cell morphology. With the combination, the expression of Bax and Bc1‐xS, the apoptosis‐enhancing proteins, was more up‐regulated and that of Bcl‐2 and Bcl‐xL, the apoptosis‐suppressing proteins, was more down‐regulated compared to the use of EPA or TNP‐470 alone, suggesting that their synergistic effect was due to an acceleration of apoptosis.

angiogenesis inhibitor apoptosis Bcl‐2 breast cancer eicosapentaenoic acid TNP‐470 


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  1. 1.
    Bang HO, Dyerberg J, Hjørne N: The composition of food consumed by Greenland Eskimos. Acta Med Scand 200: 69–73, 1976Google Scholar
  2. 2.
    Nielsen NH, Hansen JPH: Breast cancer in Greenland – selected epidemiological, clinical, and histological features. J Cancer Res Clin Oncol 98: 287–299, 1980Google Scholar
  3. 3.
    Kaizer L, Boyd NF, Kriukov V, Tritchler D: Fish consumption and breast cancer risk: An ecological study. Nutr Cancer 12: 61–68, 1989Google Scholar
  4. 4.
    Rose DP, Connolly JM: Effects of fatty acids and inhibitors of eicosanoid synthesis on the growth of a human breast cancer cell line in culture. Cancer Res 50: 7139–7144, 1990Google Scholar
  5. 5.
    Grammatikos SI, Subbaiah PV, Victor TA, Miller WM: n-3 and n-6 fatty acid processing and growth effects in neoplastic and non-cancerous human mammary epithelial cell lines. Br J Cancer 70: 219–227, 1994Google Scholar
  6. 6.
    Chajès V, Sattler W, Stranzl A, Kostner GM: Influence of n-3 fatty acids on the growth of human breast cancer cells in vitro: Relationship to peroxides and vitamin-E. Breast Cancer Res Treat 34: 199–212, 1995Google Scholar
  7. 7.
    Liu X-H, Rose DP: Suppression of type IV collagenase in MDA-MB-435 human breast cancer cells by eicosapentaenoic acid in vitro and in vivo. Cancer Lett 92: 21–26, 1995Google Scholar
  8. 8.
    Rose DP, Connolly JM, Coleman M: Effect of omega-3 fatty acids on the progression of metastases after the surgical excision of human breast cancer cell solid tumors growing in nude mice. Clin Cancer Res 2: 1751–1756, 1996Google Scholar
  9. 9.
    Senzaki H, Iwamoto S, Ogura E, Kiyozuka Y, Arita S, Kurebayashi J, Takada H, Hioki K, Tsubura A: Dietary effects of fatty acids on growth and metastasis of KPL-1 human breast cancer cells in vivo and in vitro. Anticancer Res 18: 1621–1628, 1998Google Scholar
  10. 10.
    Finstad HS, Kolset SO, Holme JA, Wiger R, Farrants A-KÖ, Blomhoff R, Drevon CA: Effect of n-3 and n-6 fatty acids on proliferation and differentiation of promyelocytic leukemic HL-60 cells. Blood 84: 3799–3809, 1994Google Scholar
  11. 11.
    Lai P, Ross J, Fearon KCH, Erson JD, Carter DC: Cell cycle arrest and induction of apoptosis in pancreatic cancer cells exposed to eicosapentaenoic acid in vitro. Br J Cancer 74: 1375–1383, 1996Google Scholar
  12. 12.
    Ingber D, Fujita T, Kishimoto S, Sudo K, Kanamaru T, Brem H, Folkman J: Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth. Nature 348: 555–557, 1990Google Scholar
  13. 13.
    Kusaka M, Sudo K, Matsutani E, Kozai Y, Marui S, Fujita T, Ingber D, Folkman J: Cytostatic inhibition of endothelial cell growth by the angiogenesis inhibitor TNP-470 (AGM-1470). Br J Cancer 69: 212–216, 1994Google Scholar
  14. 14.
    Singh Y, Shikata N, Kiyozuka Y, Nambu H, Morimoto J, Kurebayashi J, Hioki K, Tsubura A: Inhibition of tumor growth and metastasis by angiogenesis inhibitor TNP-470 on breast cancer cell lines in vitro and in vivo. Breast Cancer Res Treat 45: 15–27, 1997Google Scholar
  15. 15.
    Holmgren L, O'Reilly MS, Folkman J: Dormancy of micrometastases: Balanced proliferation and apoptosis in the presence of angiogenesis suppression. Nature Med 1: 149–153, 1995Google Scholar
  16. 16.
    Figg WD, Pluda JM, Lush RM, Saville MW, Wyvill K, Reed E, Yarchoan R: The pharmacokinetics of TNP-470: A new angiogenesis inhibitor. Pharmacotherapy 17: 91–97,1997Google Scholar
  17. 17.
    Farrow SN, Brown R: New members of the Bcl-2 family and their protein partners. Curr Opin Genet Dev 6: 45–49, 1996Google Scholar
  18. 18.
    Zha H, Aimé-Sempé C, Sato T, Reed JC: Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. J Biol Chem 271: 7440–7444, 1996Google Scholar
  19. 19.
    Singh Y, Nambu H, Yoshizawa K, Hatano T, Hioki K, Tsubura A: Factors related to axillary lymph node metastasis in T1 breast carcinoma. Oncol Rep 5: 459–462, 1998Google Scholar
  20. 20.
    Bargou RC, Daniel PT, Mapara MY, Bommert K, Wagener C, Kallinich B, Royer HD, Dörken B: Expression of the Bcl-2 gene family in normal and malignant breast tissue: Low Bax-α expression in tumor cells correlates with resistance towards apoptosis. Int J Cancer 60: 854–859, 1995Google Scholar
  21. 21.
    Krajewski S, Blomqvist C, Franssila K, Krajewska M, Wasenius V-M, Niskanen F, Nordling S, Reed JC: Reduced expression of proapoptotic gene Bax is associated with poor response rates to combination chemotherapy and shorter survival in women with metastatic breast adenocarcinoma. Cancer Res 55: 4471–4478, 1995Google Scholar
  22. 22.
    Wang TTY, Phang JM: Effects of estrogen on apoptotic pathways in human breast cancer cell line MCF-7. Cancer Res 55: 2487–2489, 1995Google Scholar
  23. 23.
    Sumantran VN, Ealovega MW, Nuñez G, Clarke MF, Wicha MS: Overexpression of Bcl-xS sensitizes MCF-7 cells to chemotherapy-induced apoptosis. Cancer Res 55: 2507–2510, 1995Google Scholar
  24. 24.
    Sakakura C, Sweeney EA, Shirahama T, Igarashi Y, Hakomori S-I, Tsujimoto H, Imanishi T, Ogaki M, Ohyama T, Yamazaki J, Hagiwara A, Yamaguchi T, Sawai K, Takahashi T: Overexpression of Bax sensitizes breast cancer MCF-7 cells to cisplatin and etoposide. Surg Today 27: 676–679, 1997Google Scholar
  25. 25.
    Cailleau R, Young R, Olivé M, Reeves WJJR: Breast tumor cell lines from pleural effusions. J Natl Cancer Inst 53: 661–674, 1974Google Scholar
  26. 26.
    Keydar I, Chen L, Karby S, Weiss FR, Delarea J, Radu M, Chaitcik S, Brenner HJ: Establishment and characterization of a cell line of human breast carcinoma origin. Europ J Cancer 15: 659–670, 1979Google Scholar
  27. 27.
    Soule HD, Vazquez J, Long A, Albert S, Brennan M: A human cell line from a pleural effusion derived from a breast carcinoma. J Natl Cancer Inst 51: 1409–1413, 1973Google Scholar
  28. 28.
    Kurebayashi J, Kurosumi M, Sonoo H: A new human breast cancer cell line, KPL-1, secretes tumor-associated antigens and grows rapidly in female athymic nude mice. Br J Cancer 71: 845–853, 1995Google Scholar
  29. 29.
    Mcleskey SW, Zhang L, Kharbanda S, Kurebayashi J, Lippman ME, Dickson RB, Kern FG: Fibroblast growth factor overexpressing breast carcinoma cells as models of angiogenesis and metastasis. Breast Cancer Res Treat 39: 103–117, 1996Google Scholar
  30. 30.
    Chou T-C: The median-effect principle and the combination index for quantitation of synergism and antagonism. In: Synergism and Antagonism in Chemotherapy. Academic Press, San Diego, 1991a, pp 61–102Google Scholar
  31. 31.
    Chou JH: Quantitation of synergism and antagonism of two or more drugs by computerized analysis. In: Synergism and Antagonism in Chemotherapy. Academic Press, San Diego, 1991b, pp 223–244Google Scholar
  32. 32.
    Nambu H, Yoshizawa K, Yang J, Daigo Y, Tsubura A: Age-specific and dose-dependent retinal dysplasia and degeneration induced by a single intraperitoneal administration of NMethyl-N-Nitrosourea to rats. J Toxicol Pathol 11: 127–131, 1998Google Scholar
  33. 33.
    Koshiji M, Adachi Y, Taketani S, Takeuchi K, Hioki K, Ikehara S: Mechanisms underlying apoptosis induced by combination of 5-fluorouracil and interferon-g. Biochem Biophys Res Commun 240: 376–381, 1997Google Scholar
  34. 34.
    Bégin ME, Ells G, Das UN, Horrobin DF: Differential killing of human carcinoma cells supplemented with n-3 and n-6 polyunsaturated fatty acids. J Natl Cancer Inst 77: 1053–1062, 1986Google Scholar
  35. 35.
    Iwamoto S, Senzaki H, Kiyozuka Y, Ogura E, Takada H, Hioki K, Tsubura A: Effects of fatty acids on liver metastasis of ACL-15 rat colon cancer cells. Nutr Cancer 3l: 143–150, 1998Google Scholar
  36. 36.
    Satoh H, Ishikawa H, Fujimoto M, Fujiwara M, Yamashita YT, Yazawa T, Ohtsuki M, Hasegawa S, Kamma H: Combined effects of TNP-470 and taxol in human non-small cell lung cancer cell lines. Anticancer Res 18: 1027–1030, 1998Google Scholar
  37. 37.
    Satoh H, Ishikawa H, Fujimoto M, Fujiwara M, Yamashita YT, Yazawa T, Ohtsuki M, Hasegawa S, Kamma H: Angiocytotoxic therapy in human non-small cell lung cancer cell lines. Acta Oncol 37: 85–90, 1998Google Scholar
  38. 38.
    Arends MJ, Wyllie AH: Apoptosis: Mechanisms and roles in pathology. Int Rev Exp Pathol 32: 223–256, 1991Google Scholar
  39. 39.
    Cohen JJ: Programmed cell death in the immune system. Adv Immunol 50: 55–85, 1991Google Scholar
  40. 40.
    Bryson GJ, Harmon BV, Collins RI: A flow cytometric study of cell death: failure of some models to correlate with morphological assessment. Immunol Cell Biol 72: 35–41, 1994Google Scholar
  41. 41.
    Telford WG, King LE, Fraker PJ: Rapid quantitation of apoptosis in pure and heterogeneous cell populations using flow cytometry. J Immunol Methods 172: 1–16, 1994Google Scholar
  42. 42.
    Calviello G, Palozza P, Piccioni E, Maggiano N, Frattucci A, Franceschelli P, Bartoli GM: Dietary supplementation with eicosapentaenoic and docosahexaenoic acid inhibits growth of Morris hepatocarcinoma 03924A in rats: Effects on proliferation and apoptosis. Int J Cancer 75: 699–705, 1998Google Scholar
  43. 43.
    Boise LH, González-GarcÍa M, Postema CE, Ding L, Lindsten T, Turka LA, Mao X, Nuñez G, Thompson CB: Bcl-x: A Bcl-2-related gene that functions as a dominant regulator of apoptotic cell death. Cell 74: 597–608, 1993Google Scholar
  44. 44.
    Oltavai ZN, Milliman CL, Korsmeyer SJ: Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell 74: 609–619, 1993Google Scholar
  45. 45.
    Yang E, Korsmeyer SJ: Molecular thanatopsis: A discourse on the Bcl 2 family and cell death. Blood 88: 386–401, 1996Google Scholar

Copyright information

© Kluwer Academic Publishers 1999

Authors and Affiliations

  • Daigo Yamamoto
    • 1
  • Yasuhiko Kiyozuka
    • 1
  • Yasushi Adachi
    • 1
  • Hideho Takada
    • 2
  • Koshiro Hioki
    • 2
  • Airo Tsubura
    • 1
  1. 1.Department of Pathology IIKansai Medical UniversityMoriguchi, OsakaJapan
  2. 2.Department of Surgery IIKansai Medical UniversityMoriguchi, OsakaJapan

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