Conjugated docosahexaenoic acid suppresses KPL-1 human breast cancer cell growth in vitro and in vivo: potential mechanisms of action

Breast Cancer Research

Airo Tsubura³  Yutaka Ogawa¹  Teruo Miyazawa⁴  Takahiko Hada⁵  Hideho Takada²  Norihisa Uehara³  Yasuhiko Kiyozuka³  Hideto Senzaki³  Naoyuki Danbara³  Takashi Yuri³  Miki Tsujita-Kyutoku¹ 

[1] Department of Plastic and Reconstructive Surgery, Kansai Medical University, Moriguchi, Osaka, Japan;Division of Surgery, Kansai Medical University Kori Hospital, Neyagawa, Osaka, Japan;Department of Pathology II, Kansai Medical University, Moriguchi, Osaka, Japan;Laboratory of Biodynamic Chemistry, Tohoku University Graduate School of Life Science and Agriculture, Sendai, Japan;R&D Division, Bizen Chemical Co., Ltd, Okayama, Japan

关键词: human;    docosahexaenoic acid;    conjugated docosahexaenoic acid;    breast cancer;    apoptosis;   

Others  :  1118753
DOI  :  10.1186/bcr789

 received in 2003-12-17, accepted in 2004-03-12,  发布年份 2004

【 摘 要 】

Introduction

The present study was conducted to examine the effect of conjugated docosahexaenoic acid (CDHA) on cell growth, cell cycle progression, mode of cell death, and expression of cell cycle regulatory and/or apoptosis-related proteins in KPL-1 human breast cancer cell line. This effect of CDHA was compared with that of docosahexaenoic acid (DHA).

Methods

KPL-1 cell growth was assessed by colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay; cell cycle progression and mode of cell death were examined by flow cytometry; and levels of expression of p53, p21^Cip1/Waf1, cyclin D₁, Bax, and Bcl-2 proteins were examined by Western blotting analysis. In vivo tumor growth was examined by injecting KPL-1 cells subcutaneously into the area of the right thoracic mammary fat pad of female athymic mice fed a CDHA diet.

Results

CDHA inhibited KPL-1 cells more effectively than did DHA (50% inhibitory concentration for 72 hours: 97 μmol/l and 270 μmol/l, respectively). With both CDHA and DHA growth inhibition was due to apoptosis, as indicated by the appearance of a sub-G₁ fraction. The apoptosis cascade involved downregulation of Bcl-2 protein; Bax expression was unchanged. Cell cycle progression was due to G₀/G₁ arrest, which involved increased expression of p53 and p21^Cip1/Waf1, and decreased expression of cyclin D₁. CDHA modulated cell cycle regulatory proteins and apoptosis-related proteins in a manner similar to that of parent DHA. In the athymic mouse system 1.0% dietary CDHA, but not 0.2%, significantly suppressed growth of KPL-1 tumor cells; CDHA tended to decrease regional lymph node metastasis in a dose dependent manner.

Conclusion

CDHA inhibited growth of KPL-1 human breast cancer cells in vitro more effectively than did DHA. The mechanisms of action involved modulation of apoptosis cascade and cell cycle progression. Dietary CDHA at 1.0% suppressed KPL-1 cell growth in the athymic mouse system.

【 授权许可】

   
2004 Tsujita-Kyutoku et al.; licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]Doll R, Peto R: The causes of cancer: quantitative estimates of avoidable risks of cancer in the United States today. J Natl Cancer Inst 1981, 66:1191-1308.
• [2]Hunter DJ, Spiegelman D, Adami HO, Beeson L, van den Brandt PA, Folsom AR, Fraser GE, Goldbohm RA, Graham S, Howe GR, Kushi LH, Marshall JR, McDermott A, Miller AB, Speizer FE, Wolk A, Yaun S-S, Willett W: Cohort studies of fat intake and the risk of breast cancer: a pooled analysis. N Engl J Med 1996, 334:356-361.
• [3]Bartsch H, Nair J, Owen RW: Dietary polyunsaturated fatty acids and cancers of the breast and colorectum: emerging evidence for their role as risk modifiers. Carcinogenesis 1999, 20:2209-2218.
• [4]Bang HO, Dyerberg J, Hjoorne N: The composition of food consumed by Greenland Eskimos. Acta Med Scand 1976, 200:69-73.
• [5]Nobmann ED, Byers T, Lanier AP, Hankin JH, Jackson MY: The diet of Alaska Native adults: 1987–1988. Am J Clin Nutr 1992, 55:1024-1032.
• [6]Kaizer L, Boyd NF, Kriukov V, Tritcher D: Fish consumption and breast cancer risk: an ecological study. Nutr Cancer 1989, 12:61-68.
• [7]Rose DP, Connolly JM, Coleman M: Effect of omega-3 fatty acids on the progression of metastasis after the surgical excision of human breast cancer cell solid tumor growing in nude mice. Clin Cancer Res 1996, 2:1751-1756.
• [8]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 1998, 18:1621-1628.
• [9]Hirose M, Masuda A, Ito N, Kamano K, Okuyama H: Effect of dietary perilla oil, soybean oil and safflower oil on 7,12-dimethylbenzanthracene (DMBA) and 1,2-dimethylhydrazine (DMH)-induced mammary gland and colon carcinogenesis in female SD rats. Carcinogenesis 1990, 11:731-735.
• [10]Hirose M, Nishikawa A, Shibutani M, Imai T, Shirai T: Chemoprevention of heterocyclic amine-induced mammary carcinogenesis in rats. Environ Mol Mutagen 2002, 39:271-278.
• [11]Ha YL, Grimm NK, Pariza MW: Anticarcinogens from fried ground beef: heat-altered derivatives of linoleic acid. Carcinogenesis 1987, 8:1881-1887.
• [12]MacDonald HB: Conjugated linoleic acid and disease prevention: a review of current knowledge. J Am Coll Nutr 2000, Suppl:111S-118S.
• [13]Kelly GS: Conjugated linoleic acid: a review. Altern Med Rev 2001, 6:367-382.
• [14]Pariza MW, Park Y, Cook ME: The biologically active isomers of conjugated linoleic acid. Prog Lipid Res 2001, 40:283-298.
• [15]Ip C, Chin SF, Scimeca JA, Pariza MW: Mammary cancer prevention by conjugated dienoic derivative of linoleic acid. Cancer Res 1991, 51:6118-6124.
• [16]Thompson H, Zhu Z, Banni S, Darcy K, Loftus T, Ip C: Morphological and biochemical status of mammary gland as influenced by conjugated linoleic acid: implication for a reduction in mammary cancer risk. Cancer Res 1997, 57:5067-5072.
• [17]Ip C, Banni S, Angioni E, Carta G, McGinly J, Thompson HJ, Barbano D, Bauman D: Conjugated linoleic acid-enriched butter fat alters mammary gland morphogenesis and reduces cancer risk in rats. J Nutr 1999, 129:2135-2142.
• [18]O'Shea M, Devery R, Lawless F, Murphy J, Stanton C: Milk fat conjugated linoleic acid (CLA) inhibits growth of human mammary MCF-7 cancer cells. Anticancer Res 2000, 20:3591-3601.
• [19]Yuri T, Danbara N, Tsujita-Kyutoku M, Fukunaga K, Takada H, Inoue Y, Hada T, Tsubura A: Dietary docosahexaenoic acid suppresses N-methyl-N-nitrosourea-induced mammary carcinogenesis in rats more effectively than eicosapentaenoic acid. Nutr Cancer 2003, 45:211-217.
• [20]Lopez A, Gerwick WH: Two new icosapentaenoic acids from the temperate red seaweed Ptilota filicina J. Agardh. Lipids 1987, 22:190-194.
• [21]Igarashi M, Miyazawa T: Do conjugated eicosapentaenoic acid and conjugated docosahexaenoic acid induce apoptosis via lipid peroxidation in cultured human tumor cells? Biochem Biophys Res Commun 2000, 270:649-656.
• [22]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 1995, 71:845-853.
• [23]Nakagawa H, Tsuta K, Kiuchi K, Senzaki H, Tanaka K, Hioki K, Tsubura A: Growth inhibitory effects of diallyl disulfide on human breast cancer cell lines. Carcinogenesis 2001, 22:891-897.
• [24]Bieri JG, Stoewsand GS, Briggs GM, Phillips RW, Woodard JC, Knapka JJ: Report of the American Institute of Nutrition ad hoc Committee on Standards for Nutritional Studies. J Nutr 1977, 107:1340-1348.
• [25]Geran RI, Greenberg NH, McDonald MM, Schumacher AM, Abbott BJ: Protocol for screening chemical agents and natural products against animal tumors and other biological systems. Cancer Chemother Rep 1972, 3:1-103.
• [26]Mizota A, Sato E, Taniai M, Adachi-Usami E, Nishikawa M: Protective effects of dietary docosahexaenoic acid against kainate-induced retinal degeneration in rats. Invest Ophthalmol Vis Sci 2001, 42:216-221.
• [27]Igarashi M, Miyazawa T: Newly recognized cytotoxic effect of conjugated trienoic fatty acids on cultured human tumor cells. Cancer Lett 2000, 148:173-179.
• [28]Cornelius AS, Yerram NR, Kratz DA, Spector AA: Cytotoxic effect of cis-parinaric acid in cultured malignant cells. Cancer Res 1991, 51:6025-6030.
• [29]Suzuki R, Noguchi R, Ota T, Abe M, Miyashita K, Kawada T: Cytotoxic effect of conjugated trienoic fatty acids on mouse tumor and human monocytic leukemia cells. Lipids 2001, 36:477-482.
• [30]Takagi T, Itabashi Y: Occurrence of mixtures of geometrical isomers of conjugated octadecatrienoic acids in some seed oils: analysis by open-tubular gas liquid chromatography and high performance liquid chromatography. Lipids 1981, 16:546-551.
• [31]Connolly JM, Gilhooly EM, Rose DP: Effects of reduced dietary linoleic acid intake, alone or combined with an algal source of docosahexaenoic acid, on MDA-MB-231 breast cancer cell growth and apoptosis in nude mice. Nutr Cancer 1999, 35:44-49.
• [32]Istfan NW, Wan J, Chen ZY: Fish oil and cell proliferation kinetics in a mammary carcinoma tumor model. Adv Exp Med Biol 1995, 375:149-156.
• [33]Chen ZY, Istfan NW: Docosahexaenoic acid, a major constituent of fish oil diets, prevents activation of cyclin-dependent kinases and S-phase entry by serum stimulation in HT-29 cells. Prostaglandins Leukot Essent Fatty Acids 2001, 64:67-73.
• [34]Ip C, Scimeca JA, Thompson HJ: Conjugated linoleic acid. A powerful anticarcinogen from animal fat sources. Cancer 1994, 74:1050-1054.
• [35]Futakuchi M, Cheng JL, Hirose M, Kimoto N, Cho Y-M, Iwata T, Kasai M, Tokudome S, Shirai T: Inhibition of conjugated fatty acids derived from safflower or perilla oil of induction and development of mammary tumors in rats induced by 2-amino-1-methyl-6-phenylimidazo (4,5-b) pyridine (PhIP). Cancer Lett 2002, 178:131-139.
• [36]Takata T, Minoura T, Takada H, Sakaguchi M, Yamamura M, Hioki K, Yamamoto M: Specific inhibitory effect of dietary eicosapentaenoic acid on N-nitroso-N-methylurea-induced mammary carcinogenesis in female Sprague–Dawley rats. Carcinogenesis 1990, 11:2015-2019.