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Dietary supplementation with secoisolariciresinol diglycoside (SDG) reduces experimental metastasis of melanoma cells in mice
Cancer Letters 142 (1999) 91±96
Dietary supplementation with secoisolariciresinol diglycoside (SDG) reduces experimental metastasis of melanoma cells in mice Donghua Li a, John A. Yee a, Lilian U. Thompson b, Lin Yan a,* b
a Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68124-0405, USA Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ont. M5S 1A8, Canada
Received 4 December 1998; received in revised form 30 March 1999; accepted 5 April 1999
Abstract We investigated the effect of dietary supplementation with secoisolariciresinol diglycoside (SDG), a lignan precursor isolated from ¯axseed, on experimental metastasis of B16BL6 murine melanoma cells in C57BL/6 mice. Four diets were compared: a basal diet (control group) and the basal diet supplemented with SDG at 73, 147 or 293 mmol/kg (equivalent to SDG provided in the 2.5, 5 or 10% ¯axseed diet). Mice were fed the diet for 2 weeks before and after an intravenous injection of 0:6 £ 105 tumor cells. At necropsy, the number and size of tumors that formed in the lungs were determined. The median number of tumors in the control group was 62, and those in the SDG-supplemented groups were 38, 36 and 29, respectively. The last was signi®cantly different from the control (P , 0:01). Dietary supplementation with SDG at 73, 147 and 293 mmol/kg also decreased tumor size (tumor cross-sectional area and volume) in a dose-dependent manner compared with the control values. These results show that SDG reduced pulmonary metastasis of melanoma cells and inhibited the growth of metastatic tumors that formed in the lungs. It is concluded that dietary supplementation with SDG reduces experimental metastasis of melanoma cells in mice. q 1999 Published by Elsevier Science Ltd. All rights reserved. Keywords: Lignans; Diet; Mice; Melanoma; Metastasis
1. Introduction Cancer incidence, particularly of the breast, prostate and colon, is lower in Asian countries than that in the West [1]. Diet is an important environmental factor that has been associated with the difference in cancer risk among countries. It has long been suggested that consumption of foods that are from plant sources protects humans against cancer because of their high ®ber and low fat content. However, recent research suggests that other constituents of * Corresponding author. Tel.: 1 1-402-280-1207; fax: 1 1-402280-2690. E-mail address: [email protected] (L. Yan)
plant foods, e.g. phytoestrogens, may also account for their anticancer effect. Lignans are a major group of dietary phytoestrogens. The primary mammalian lignans are enterodiol and enterolactone that are formed from their plant lignan precursors, secoisolariciresinol and matairesinol, respectively, by the action of bowel micro¯ora. Mammalian lignan precursors are found in oilseeds, whole grains, legumes and certain vegetables, but ¯axseed is the richest source [2]. Thus, ¯axseed has been used as a source of lignans to investigate the anticancer effect of dietary lignans in animal studies. Consumption of foods that are high in dietary phytoestrogens, including lignans, has been correlated with a decreased risk of breast cancer in humans [3].
0304-3835/99/$ - see front matter q 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 0304-383 5(99)00158-5
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D. Li et al. / Cancer Letters 142 (1999) 91±96
Laboratory studies show that dietary supplementation with ¯axseed reduces chemically induced mammary and colon tumorigenesis in rats [4±6]. Supplementation of secoisolariciresinol diglycoside (SDG), a mammalian lignan precursor isolated from ¯axseed, to the animal diet has a similar inhibitory effects on mammary and colon carcinogenesis [6±8]. Many biological properties of lignans seen in in vitro studies may be responsible for their anticancer properties. These include estrogenic and antiestrogenic [9], antioxidative [10,11], antiproliferative [12,13], and antiaromatase activities [14]. We recently investigated the effect of dietary ¯axseed in the prevention of metastasis. Metastasis, the spread of malignant cells from a primary tumor to distant organs that results in the development of secondary tumors, is the most devastating aspect of cancer. Advances in surgery and other therapeutic approaches have signi®cantly improved the treatment of primary tumors. However, metastasis remains a major cause of poor prognosis and death in cancer patients. We found that dietary supplementation with ¯axseed reduces experimental metastasis of melanoma cells in mice when tumor cells were intravenously injected into animals [15]. These results indicate that dietary intake of ¯axseed may be bene®cial in preventing the spread of malignant cells. The objective of the present study was to determine whether lignans present in ¯axseed reduce metastasis. The effect of dietary supplementation with SDG on pulmonary metastasis of melanoma cells in mice was investigated using an intravenous injection model.
2. Materials and methods 2.1. Animals and diet The protocol of the present study was reviewed and approved by the Creighton University Animal Care and Use Committee and complied with the Guide for the Care and Use of Laboratory Animals [16]. Male C57BL/6 mice (3 weeks old) were purchased from Charles River (Wilmington, MA). Mice were housed ®ve per box, in wire-topped plastic boxes, in a pathogen-free room on a 12:12 h light±dark cycle. The temperature in the room was maintained at 25 ^ 18C. Mice were given free access to the diet and deio-
nized water and weighed weekly. Four diets were compared: a basal diet (control group) and the basal diet supplemented with SDG at 73, 147, or 293 mmol/kg, which was equivalent to that provided in the 2.5, 5 or 10% ¯axseed diet [15]. The SDG was isolated from ¯axseed as described previously [8]. Dietary formulations were based on the AIN-93G diet recommended by the American Institute of Nutrition [17], except that soybean oil was replaced with corn oil. Diet components were purchased from ICN (Costa Mesa, CA). All diets were prepared in our laboratory, and each lot was stored at 48C for no longer than 3 weeks. 2.2. Experimental design Mice were fed the basal diet for 2 days before being randomly assigned to four groups (27±28 mice per group). They were then fed the basal diet or the SDG-supplemented diets. B16BL6 murine melanoma cells (Dr. I.J. Fidler, University of Texas, Houston, TX) were cultured in minimum essential medium with 10% heat-inactivated fetal bovine serum as described previously [15]. The melanoma cells were collected from monolayer cultures by a brief trypsinization (0.05% trypsin and 0.53 mM EDTA). The viability of the cells was determined with Trypan Blue, and a single cell suspension was made in serum-free medium. After 2 weeks on the diets, each mouse was injected with 0:6 £ 105 viable cells in 0.2 ml via the lateral tail vein. To avoid possible changes in cell viability, melanoma cells were injected into mice within 30 min after their collection. The order that tumor cells were injected into mice from different dietary groups was randomized. The mice were then fed the diets for another 2 weeks. Six mice from each group were transferred to metabolic cages for 1 week before tumor cell injection, and their food intake was recorded. Urine collected from each group throughout this week was pooled and quanti®ed for lignans (i.e. enterolactone and enterodiol) using a gas chromatography±mass spectrometry procedure [2,8]. The SDG intake was calculated on the basis of food intake and the dietary SDG concentration. At the end of the experiment, mice were anesthetized using ketamine (50 mg/kg body weight) and xylazine (5 mg/kg body weight) and then terminated
D. Li et al. / Cancer Letters 142 (1999) 91±96
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Table 1 Effect of dietary supplementation with SDG on pulmonary metastasis of melanoma cells in mice a Group
Control SDG 73 mmol/kg 147 mmol/kg 293 mmol/kg
Mice
Mice with lung tumors
Tumors/mouse
n
1±50 Tumors
.50 Tumors b
Median c
Mean ^ SE
Range
27
11
16
62
64 ^ 8
10±180
27 28 27
19 22 21
8 6b 6b
38 36 29 c
43 ^ 5 42 ^ 4 33 ^ 4
8±117 9±96 4±86
a
The method used to determine the number of lung tumors is described in Section 2. Because of the heterogeneous variances among sample populations, the mean values were not analyzed by ANOVA. b Signi®cantly different from the control, P # 0:05. Data were analyzed using Fisher's exact test. c Signi®cantly different from the control, P # 0:01. Data were analyzed using the Kruskal±Wallis non-parametric and Dunn's multiple comparison tests.
by cervical dislocation. Their lungs were excised and ®xed in 10% phosphate-buffered formalin. The number of pulmonary tumors was determined by counting the visible black foci using a dissecting microscope [15]. The cross-sectional area of tumors in randomly selected ®elds from the lungs of tumorbearing mice was measured using a Quantimet 500 image analysis system (Leica Cambridge, Cambridge, UK). The tumor volume was calculated using the mean of the longest and the shortest diameters measured and the assumption that tumors were spherical [18]. 2.3. Statistical analysis Fisher's exact test was used to analyze the frequency distribution of the mice that had 1±50 tumors or .50 tumors. Bartlett's test for homogeneity of variances revealed that the standard deviations for the mean values of the number of lung tumors, tumor cross-sectional area, and tumor volume differed signi®cantly among the groups (P , 0:05). Since only means with homogeneous variances can be analyzed by ANOVA, the median values were compared using the Kruskal±Wallis non-parametric and Dunn's multiple comparison tests. Differences were considered signi®cant at P # 0:05. 3. Results Our results show that dietary supplementation with SDG at concentrations used in the present study had
no adverse effect on the growth of mice during the experimental period. The mean body weight of all mice at the beginning and at the end of the experiment was 12 ^ 2 and 22 ^ 1 g, respectively. There were no differences in body weight among the groups throughout the experiment (data not shown). Liver, kidneys, spleen and heart were collected and weighed at the end of the experiment, and there was no signi®cant difference in organ weights between the groups (data not shown). The mean food intake of all mice was 2:4 ^ 0:4 g/day, and there were no differences among the dietary groups (data not shown). The daily SDG intake was 0, 0.18, 0.37 and 0.73 mmol in mice fed the basal diet and the diet supplemented with SDG at 73, 147 and 293 mmol/kg, respectively. The urinary excretion of lignans (a sum of enterolactone and enterodiol) was 0, 115, 226 and 343 nmol per mouse per day in mice fed the basal diet and the SDG-supplemented diets at concentrations described above. Intravenous injection of B16 melanoma cells into C57BL/6 mice results in pulmonary metastasis [15,19]. Injection of 0:6 £ 105 viable B16BL6 cells via the lateral tail vein resulted in lung tumor colonies in all the mice used in the present study (Table 1). Based on the number of lung tumors per animal, the mice were placed into one of the two categories: (1) 1±50 tumors and (2) .50 tumors. In the control group, 16 of the 27 (59%) mice had .50 lung tumors (Table 1). In contrast, 30, 21 and 22% of the mice in groups fed the diet containing SDG at 73, 147 and 293 mmol/kg had .50 tumors. The latter two
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D. Li et al. / Cancer Letters 142 (1999) 91±96
Table 2 Effect of dietary supplementation with SDG on tumor cross-sectional area and volume of metastatic tumors that developed in the lungs a Group
Control SDG 73 mmol/kg 147 mmol/kg 293 mmol/kg
Tumors
Tumor cross-sectional area (mm 2)
Tumor volume (mm 3)
n
Median b
Mean ^ SE
Median b
Mean ^ SE
211
0.20
0.25 ^ 0.01
0.09
0.17 ^ 0.01
211 211 211
0.09 0.08 b 0.06 b
0.18 ^ 0.01 0.15 ^ 0.01 0.13 ^ 0.01
0.06 0.03 b 0.01 b
0.12 ^ 0.01 0.11 ^ 0.01 0.04 ^ 0.01
a
The methods used to determine the tumor cross-sectional area and volume are described in Section 2. Because of the heterogeneous variances among sample populations, the mean values were not analyzed by ANOVA. b Signi®cantly different from the control, P # 0:01. Data were analyzed using the Kruskal±Wallis non-parametric and Dunn's multiple comparison tests.
were signi®cantly different from the control (P # 0:05). The difference in the number of lung tumors between the control group and the SDGsupplemented groups was compared. The median number of lung tumors in the control group was 62, and that in the SDG-treated groups was 38, 36 and 29 (Table 1). The last was signi®cantly different from the control (P # 0:01). The mean number of lung tumors in the control group was 64 ^ 8, and that in groups fed the SDG-supplemented diets was 33, 34 and 48% lower than the control group, respectively. To determine the effect of SDG on the size of metastatic tumors that formed in the lungs, tumor crosssectional area and volume were measured [15,18]. As shown in Table 2, the median cross-sectional area was 0.20 mm 2, and the median volume was 0.09 mm 3 in mice fed the basal diet. Supplementation of SDG to the diets reduced these variables in a dose-dependent manner compared with the controls. The difference between the control group and the groups with SDG at 147 and 293 mmol/kg was signi®cant (P # 0:01). Dietary supplementation with SDG also decreased the mean values of tumor cross-sectional area and volume in a dose-dependent manner. 4. Discussion The results of the present study demonstrate that dietary supplementation with SDG at concentrations equivalent to that provided in the ¯axseed diets [15] reduced the number of lung tumors and tumor crosssectional area and volume compared with the controls.
The dietary content of SDG was positively correlated with both urinary concentration of lignans and the magnitude of the inhibition on metastasis. These results indicate that SDG or mammalian lignans derived from SDG effectively reduced pulmonary metastasis of melanoma cells in mice and retarded the growth of metastatic tumors that developed in the lungs. This supports our previous ®nding that dietary supplementation with ¯axseed reduces experimental metastasis [15], and identi®es SDG as an active antimetastatic constituent of ¯axseed. Results from this study provide the ®rst evidence that dietary lignans reduce experimental metastasis. To metastasize, a malignant cell or a group of cells leave the primary tumor, invade the local tissue, intravasate into the blood circulatory system, arrest in a vascular bed of the target organ, extravasate into the interstitium of the organ, and proliferate to form metastatic tumors. The process can take place once again to yield other metastases. Interventions that block any of these steps can theoretically prevent the spread of malignant cells. In the intravenous injection model used in the present study, the melanoma cells were directly injected into the cardiovascular system. This model eliminates intravasation, but measures the ability of malignant cells to extravasate into the lung tissue. We found that dietary supplementation with SDG decreased the number of metastatic tumors that developed in the lungs. This suggests that SDG or mammalian lignans activated from SDG affect extravasation of malignant cells. Extravasation, as well as intravasation, is a threestep invasive process. It includes the adhesion of
D. Li et al. / Cancer Letters 142 (1999) 91±96
cancer cells to the extracellular matrix, proteolytic degradation, and migration of malignant cells through the degraded region. At present, there have been no publications on lignans and cancer invasion. However, results of in vitro studies show that iso¯avones, another major group of dietary phytoestrogens, affect the invasive behavior of neoplastic cells. This includes the inhibition of cell adhesion to the extracellular matrix proteins [20] and invasion of cancer cells through a reconstituted extracellular matrix barrier [21]. Although exposure of cancer cells to iso¯avones in vitro is not comparable with providing animals iso¯avone-supplemented diets, these results indicate that dietary phytoestrogens may reduce metastasis by inhibiting invasion. Because of the structural and biological similarities of lignans to iso¯avones, the effect of lignans on cancer invasion deserves further investigation. In the present study, we found that tumor crosssectional area and volume of mice fed the SDG diets were decreased in a dose-dependent manner compared with those of mice fed the basal diet. A decrease in tumor size could be due to prolonged retention of tumor cells in the circulatory system or an inhibition of malignant cell proliferation after they take up residence in the lungs. Quantitative analysis on experimental metastasis of B16 melanoma cells has shown that most circulating tumor cells die following injection [19,22]. Approximately 1% of the cells survives for 24 h, and one tenth of them form lung colonies. Therefore, it seems unlikely that retention in the circulation is responsible for the difference in tumor size between the SDG-supplemented groups and the control group. It is more likely that this difference is due to the inhibition of mitosis of malignant cells in the lungs. In vitro experiments show that mammalian lignans inhibit proliferation of tumor cells [12,13]. Flaxseed and SDG inhibit the growth of chemically induced mammary tumors when they are provided to rats after the establishment of the primary tumor [7]. Our results are in agreement with the previous publication that dietary lignans inhibit the growth of the primary tumors [7]. We suggest that lignans also inhibit the growth of the metastatic tumors. Early epidemiologic investigations demonstrate that Japanese breast cancer patients have a higher survival rate and a better prognosis than their Western
95
counterparts [23,24]. Japanese patients are less likely to have histologic evidence of local invasion and regional metastasis than American patients at the time of diagnosis or surgery [25,26]. Although these studies were not designed to correlate the nutritional status of patients with the disease risk, it is unlikely that the differences can be solely explained by ethnicity. Rather, environmental differences between the two populations, particularly in the diet, may be important contributors. A previous clinical trial showed that dietary supplementation with a combination of nutritional antioxidants, in addition to surgical and other therapeutic treatments, prevents further metastasis in breast cancer patients with tumor spread to lymph nodes in axilla [27]. Taken together, these results suggest that dietary habits and nutritional intervention affect the prognosis of post-surgery cancer patients. We have shown that dietary supplementation with soybean [28] and ¯axseed [15] reduce experimental metastasis. A similar inhibitory effect on experimental metastasis was observed when iso¯avones genistein and daidzein were supplemented to the animal diets [29]. The present study demonstrated that dietary supplementation with SDG protects mice against pulmonary metastasis of melanoma cells. Based on these results, we suggest that dietary phytoestrogens can be used as nutritional adjuvants in preventing metastasis in human cancer patients. In summary, the results of the present study demonstrate that dietary supplementation with SDG reduced experimental metastasis of melanoma cells in mice and also inhibited the growth of metastatic tumors that developed in the lungs. We conclude that SDG or mammalian lignans activated from SDG are responsible, at least in part, for the protective effect of dietary ¯axseed on experimental metastasis of melanoma cells in mice. Acknowledgements The authors thank Vivian W. Huang and Mai-Linh Frascarelli, undergraduate students at Creighton University, for participating in this research project, Felicia Cheung, University of Toronto, for technical assistance with the lignan analysis, and Dr. Gleb R. Haynatzki who provided help with statistical analysis
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of the data. This work was supported by the Creighton Health Future Foundation. References [1] American Cancer Society, Facts and Figs., American Cancer Society, (1994) Atlanta, GA. [2] L.U. Thompson, P. Robb, M. Serraino, F. Cheung, Mammalian lignan production from various foods, Nutr. Cancer 16 (1991) 43±52. [3] D. Ingram, K. Sanders, M. Kolybaba, D. Lopez, Case-control study of phytoestrogens and breast cancer, Lancet 350 (1997) 990±994. [4] M. Serraino, L.U. Thompson, The effect of ¯axseed supplementation on early risk markers for mammary carcinogenesis, Cancer Lett. 60 (1991) 135±142. [5] M. Serraino, L.U. Thompson, The effect of ¯axseed supplementation on the initiation and promotion stages of mammary tumorigenesis, Nutr. Cancer 17 (1992) 153±159. [6] M. Jenab, L.U. Thompson, The in¯uence of ¯axseed and lignans on colon carcinogenesis and beta-glucuronidase activity, Carcinogenesis 17 (1996) 1343±1348. [7] L.U. Thompson, S.E. Richard, L.J. Orcheson, M.M. Seidl, Flaxseed and its lignan and oil components reduce mammary tumor growth at a late stage of carcinogenesis, Carcinogenesis 17 (1996) 1373±1376. [8] L.U. Thompson, M.M. Seidl, S.E. Rickard, L.J. Orcheson, H.H. Fong, Antitumorigenic effect of a mammalian lignan precursor from ¯axseed, Nutr. Cancer 26 (1996) 159±165. [9] M.S. Kurzer, J.L. Slavin, H. Adlercreutz, Flaxseed, lignans, and sex hormones, in: S.C. Cunnane, L.U. Thompson (eds), Flaxseed in Human Nutrition, AOCS Press, Champagne, 1995, pp. 136-144. [10] R. Amarowicz, U. Wanasundara, J. Wanasundara, F. Shahidi, Antioxidant activity of ethanolic extracts of ¯axseed in a betacarotene-linoleate model system, J. Food Lipids 1 (1993) 111± 117. [11] H. Lu, G.T. Liu, Effect of dibenzo[a,c]cyclooctene lignans isolated from Fructus schizandrae on lipid peroxidation and antioxidative enzyme activity. Chem.-Biol. Interact. 78 (1991) 77±84. [12] T. Hirano, K. Fukuoka, T. Oka, T. Naito, H. Hosaka, Y. Mitsuhashi, Y. Matsumoto, Antiproliferative activity of mammalian lignan derivatives against human breast carcinoma cell line, ZR-75-1, Cancer Invest. 8 (1990) 595±602. [13] M.K. Sung, M. Lautens, L.U. Thompson, Mammalian lignans inhibits the growth of estrogen-independent human colon tumor cells, Anticancer Res. 18 (1998) 1405±1408. [14] C. Wang, T. Makela, T. Hase, H. Adlercreutz, M.S. Kurzer, Lignans and ¯avonoids inhibit aromatase enzyme in human preadipocytes, J. Steroid Biochem. Mol. Biol. 50 (1994) 205± 212. [15] L. Yan, J.A. Yee, D. Li, M.H. McGuire, L.U. Thompson,
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Dietary ¯axseed supplementation and experimental metastasis of melanoma cells in mice, Cancer Lett. 124 (1998) 181±186. National Institute of Health, Guide for the Care and Use of Laboratory Animals, National Academy Press, (1996) Washington, DC. P.G. Reeves, F.H. Nielsen, G.C. Fahey, Puri®ed diets for laboratory rodents: ®nal report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet, J. Nutr. 123 (1993) 1939±1951. D.R. Welch, A. Neri, G.L. Nicolson, Comparison of `spontaneous' and `experimental' metastasis using rat 13726 mammary adenocarcinoma metastatic cell clones, Invasion Metastasis 3 (1983) 65±80. I.J. Fidler, Biological behavior of malignant melanoma cells correlated to their survival in vivo, Cancer Res. 35 (1975) 218±224. C.E. Giovanni, G. Nicoletti, M. Sensi, A. Santoni, G. Palmieri, L. Landuzzi, P. Nanni, P.L. Lollini, H-2Kb and H-2Db gene transfections in B16 melanoma differently affect non-immunological properties relevant to the metastatic process, Involvement of integrin molecules, Int. J. Cancer 59 (1994) 269± 274. E.M. Scholar, M.L. Toews, Inhibition of invasion of murine mammary carcinoma cells by the tyrosine kinase inhibitor genistein, Cancer Lett. 87 (1994) 157±162. I.J. Fidler, Metastasis: quantitative analysis of distribution and fate of tumor emboli labeled with 125I-5-iodo-2 0 -deoxyuridine, J. Natl. Cancer Inst. 45 (1970) 773±782. A.S. Morrison, C.R. Lowe, B. MacMahon, B. Ravnihar, S. Yuasa, Some international differences in treatment and survival in breast cancer, Int. J. Cancer 18 (1976) 269±273. E.L. Wynder, T. Kajitani, J. Kuno, J.C. Lucas, A. De Palo, J. Farrow, comparison of survival rates between American and Japanese patients with breast cancer, Surg. Gynecol. Obstet. 117 (1963) 196±220. P.P. Rosen, R. Ashikari, H. Thaler, S. Ishikawa, T. Hirota, O. Abe, H. Yamamoto, E.J. Beattie, J.A. Urban, V. Mike, Comparative study of some pathologic features of mammary carcinoma in Tokyo, Japan and New York, USA, Cancer 39 (1977) 429±434. G.N. Stemmermann, A. Catts, F.H. Fukunaga, A. Horie, A.M.Y. Nomura, Breast cancer in women of Japanese and Caucasian ancestry in Hawaii, Cancer 56 (1985) 206±209. K. Lockwood, S. Moesgaard, T. Hanioka, K. Folkers, Apparent partial remission of breast cancer in `high risk' patients supplemented with nutritional antioxidants, essential fatty acids and coenzyme Q10, Mol, Aspects Med. 15 (1994) s231±s240. L. Yan, J.A. Yee, M.H. McGuire, G.L. Graef, Effect of dietary supplementation of soybeans on experimental metastasis of melanoma cells in mice, Nutr. Cancer 29 (1997) 1±6. D. Li, J.A. Yee, M.H. McGuire, L. Yan, Soybean iso¯avones reduce experimental metastasis of melanoma cells in mice, J. Nutr. (1999) in press.
Dietary supplementation with secoisolariciresinol diglycoside (SDG) reduces experimental metastasis of melanoma cells in mice Donghua Li a, John A. Yee a, Lilian U. Thompson b, Lin Yan a,* b
a Department of Biomedical Sciences, Creighton University School of Medicine, Omaha, NE 68124-0405, USA Department of Nutritional Sciences, Faculty of Medicine, University of Toronto, Toronto, Ont. M5S 1A8, Canada
Received 4 December 1998; received in revised form 30 March 1999; accepted 5 April 1999
Abstract We investigated the effect of dietary supplementation with secoisolariciresinol diglycoside (SDG), a lignan precursor isolated from ¯axseed, on experimental metastasis of B16BL6 murine melanoma cells in C57BL/6 mice. Four diets were compared: a basal diet (control group) and the basal diet supplemented with SDG at 73, 147 or 293 mmol/kg (equivalent to SDG provided in the 2.5, 5 or 10% ¯axseed diet). Mice were fed the diet for 2 weeks before and after an intravenous injection of 0:6 £ 105 tumor cells. At necropsy, the number and size of tumors that formed in the lungs were determined. The median number of tumors in the control group was 62, and those in the SDG-supplemented groups were 38, 36 and 29, respectively. The last was signi®cantly different from the control (P , 0:01). Dietary supplementation with SDG at 73, 147 and 293 mmol/kg also decreased tumor size (tumor cross-sectional area and volume) in a dose-dependent manner compared with the control values. These results show that SDG reduced pulmonary metastasis of melanoma cells and inhibited the growth of metastatic tumors that formed in the lungs. It is concluded that dietary supplementation with SDG reduces experimental metastasis of melanoma cells in mice. q 1999 Published by Elsevier Science Ltd. All rights reserved. Keywords: Lignans; Diet; Mice; Melanoma; Metastasis
1. Introduction Cancer incidence, particularly of the breast, prostate and colon, is lower in Asian countries than that in the West [1]. Diet is an important environmental factor that has been associated with the difference in cancer risk among countries. It has long been suggested that consumption of foods that are from plant sources protects humans against cancer because of their high ®ber and low fat content. However, recent research suggests that other constituents of * Corresponding author. Tel.: 1 1-402-280-1207; fax: 1 1-402280-2690. E-mail address: [email protected] (L. Yan)
plant foods, e.g. phytoestrogens, may also account for their anticancer effect. Lignans are a major group of dietary phytoestrogens. The primary mammalian lignans are enterodiol and enterolactone that are formed from their plant lignan precursors, secoisolariciresinol and matairesinol, respectively, by the action of bowel micro¯ora. Mammalian lignan precursors are found in oilseeds, whole grains, legumes and certain vegetables, but ¯axseed is the richest source [2]. Thus, ¯axseed has been used as a source of lignans to investigate the anticancer effect of dietary lignans in animal studies. Consumption of foods that are high in dietary phytoestrogens, including lignans, has been correlated with a decreased risk of breast cancer in humans [3].
0304-3835/99/$ - see front matter q 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 0304-383 5(99)00158-5
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D. Li et al. / Cancer Letters 142 (1999) 91±96
Laboratory studies show that dietary supplementation with ¯axseed reduces chemically induced mammary and colon tumorigenesis in rats [4±6]. Supplementation of secoisolariciresinol diglycoside (SDG), a mammalian lignan precursor isolated from ¯axseed, to the animal diet has a similar inhibitory effects on mammary and colon carcinogenesis [6±8]. Many biological properties of lignans seen in in vitro studies may be responsible for their anticancer properties. These include estrogenic and antiestrogenic [9], antioxidative [10,11], antiproliferative [12,13], and antiaromatase activities [14]. We recently investigated the effect of dietary ¯axseed in the prevention of metastasis. Metastasis, the spread of malignant cells from a primary tumor to distant organs that results in the development of secondary tumors, is the most devastating aspect of cancer. Advances in surgery and other therapeutic approaches have signi®cantly improved the treatment of primary tumors. However, metastasis remains a major cause of poor prognosis and death in cancer patients. We found that dietary supplementation with ¯axseed reduces experimental metastasis of melanoma cells in mice when tumor cells were intravenously injected into animals [15]. These results indicate that dietary intake of ¯axseed may be bene®cial in preventing the spread of malignant cells. The objective of the present study was to determine whether lignans present in ¯axseed reduce metastasis. The effect of dietary supplementation with SDG on pulmonary metastasis of melanoma cells in mice was investigated using an intravenous injection model.
2. Materials and methods 2.1. Animals and diet The protocol of the present study was reviewed and approved by the Creighton University Animal Care and Use Committee and complied with the Guide for the Care and Use of Laboratory Animals [16]. Male C57BL/6 mice (3 weeks old) were purchased from Charles River (Wilmington, MA). Mice were housed ®ve per box, in wire-topped plastic boxes, in a pathogen-free room on a 12:12 h light±dark cycle. The temperature in the room was maintained at 25 ^ 18C. Mice were given free access to the diet and deio-
nized water and weighed weekly. Four diets were compared: a basal diet (control group) and the basal diet supplemented with SDG at 73, 147, or 293 mmol/kg, which was equivalent to that provided in the 2.5, 5 or 10% ¯axseed diet [15]. The SDG was isolated from ¯axseed as described previously [8]. Dietary formulations were based on the AIN-93G diet recommended by the American Institute of Nutrition [17], except that soybean oil was replaced with corn oil. Diet components were purchased from ICN (Costa Mesa, CA). All diets were prepared in our laboratory, and each lot was stored at 48C for no longer than 3 weeks. 2.2. Experimental design Mice were fed the basal diet for 2 days before being randomly assigned to four groups (27±28 mice per group). They were then fed the basal diet or the SDG-supplemented diets. B16BL6 murine melanoma cells (Dr. I.J. Fidler, University of Texas, Houston, TX) were cultured in minimum essential medium with 10% heat-inactivated fetal bovine serum as described previously [15]. The melanoma cells were collected from monolayer cultures by a brief trypsinization (0.05% trypsin and 0.53 mM EDTA). The viability of the cells was determined with Trypan Blue, and a single cell suspension was made in serum-free medium. After 2 weeks on the diets, each mouse was injected with 0:6 £ 105 viable cells in 0.2 ml via the lateral tail vein. To avoid possible changes in cell viability, melanoma cells were injected into mice within 30 min after their collection. The order that tumor cells were injected into mice from different dietary groups was randomized. The mice were then fed the diets for another 2 weeks. Six mice from each group were transferred to metabolic cages for 1 week before tumor cell injection, and their food intake was recorded. Urine collected from each group throughout this week was pooled and quanti®ed for lignans (i.e. enterolactone and enterodiol) using a gas chromatography±mass spectrometry procedure [2,8]. The SDG intake was calculated on the basis of food intake and the dietary SDG concentration. At the end of the experiment, mice were anesthetized using ketamine (50 mg/kg body weight) and xylazine (5 mg/kg body weight) and then terminated
D. Li et al. / Cancer Letters 142 (1999) 91±96
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Table 1 Effect of dietary supplementation with SDG on pulmonary metastasis of melanoma cells in mice a Group
Control SDG 73 mmol/kg 147 mmol/kg 293 mmol/kg
Mice
Mice with lung tumors
Tumors/mouse
n
1±50 Tumors
.50 Tumors b
Median c
Mean ^ SE
Range
27
11
16
62
64 ^ 8
10±180
27 28 27
19 22 21
8 6b 6b
38 36 29 c
43 ^ 5 42 ^ 4 33 ^ 4
8±117 9±96 4±86
a
The method used to determine the number of lung tumors is described in Section 2. Because of the heterogeneous variances among sample populations, the mean values were not analyzed by ANOVA. b Signi®cantly different from the control, P # 0:05. Data were analyzed using Fisher's exact test. c Signi®cantly different from the control, P # 0:01. Data were analyzed using the Kruskal±Wallis non-parametric and Dunn's multiple comparison tests.
by cervical dislocation. Their lungs were excised and ®xed in 10% phosphate-buffered formalin. The number of pulmonary tumors was determined by counting the visible black foci using a dissecting microscope [15]. The cross-sectional area of tumors in randomly selected ®elds from the lungs of tumorbearing mice was measured using a Quantimet 500 image analysis system (Leica Cambridge, Cambridge, UK). The tumor volume was calculated using the mean of the longest and the shortest diameters measured and the assumption that tumors were spherical [18]. 2.3. Statistical analysis Fisher's exact test was used to analyze the frequency distribution of the mice that had 1±50 tumors or .50 tumors. Bartlett's test for homogeneity of variances revealed that the standard deviations for the mean values of the number of lung tumors, tumor cross-sectional area, and tumor volume differed signi®cantly among the groups (P , 0:05). Since only means with homogeneous variances can be analyzed by ANOVA, the median values were compared using the Kruskal±Wallis non-parametric and Dunn's multiple comparison tests. Differences were considered signi®cant at P # 0:05. 3. Results Our results show that dietary supplementation with SDG at concentrations used in the present study had
no adverse effect on the growth of mice during the experimental period. The mean body weight of all mice at the beginning and at the end of the experiment was 12 ^ 2 and 22 ^ 1 g, respectively. There were no differences in body weight among the groups throughout the experiment (data not shown). Liver, kidneys, spleen and heart were collected and weighed at the end of the experiment, and there was no signi®cant difference in organ weights between the groups (data not shown). The mean food intake of all mice was 2:4 ^ 0:4 g/day, and there were no differences among the dietary groups (data not shown). The daily SDG intake was 0, 0.18, 0.37 and 0.73 mmol in mice fed the basal diet and the diet supplemented with SDG at 73, 147 and 293 mmol/kg, respectively. The urinary excretion of lignans (a sum of enterolactone and enterodiol) was 0, 115, 226 and 343 nmol per mouse per day in mice fed the basal diet and the SDG-supplemented diets at concentrations described above. Intravenous injection of B16 melanoma cells into C57BL/6 mice results in pulmonary metastasis [15,19]. Injection of 0:6 £ 105 viable B16BL6 cells via the lateral tail vein resulted in lung tumor colonies in all the mice used in the present study (Table 1). Based on the number of lung tumors per animal, the mice were placed into one of the two categories: (1) 1±50 tumors and (2) .50 tumors. In the control group, 16 of the 27 (59%) mice had .50 lung tumors (Table 1). In contrast, 30, 21 and 22% of the mice in groups fed the diet containing SDG at 73, 147 and 293 mmol/kg had .50 tumors. The latter two
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D. Li et al. / Cancer Letters 142 (1999) 91±96
Table 2 Effect of dietary supplementation with SDG on tumor cross-sectional area and volume of metastatic tumors that developed in the lungs a Group
Control SDG 73 mmol/kg 147 mmol/kg 293 mmol/kg
Tumors
Tumor cross-sectional area (mm 2)
Tumor volume (mm 3)
n
Median b
Mean ^ SE
Median b
Mean ^ SE
211
0.20
0.25 ^ 0.01
0.09
0.17 ^ 0.01
211 211 211
0.09 0.08 b 0.06 b
0.18 ^ 0.01 0.15 ^ 0.01 0.13 ^ 0.01
0.06 0.03 b 0.01 b
0.12 ^ 0.01 0.11 ^ 0.01 0.04 ^ 0.01
a
The methods used to determine the tumor cross-sectional area and volume are described in Section 2. Because of the heterogeneous variances among sample populations, the mean values were not analyzed by ANOVA. b Signi®cantly different from the control, P # 0:01. Data were analyzed using the Kruskal±Wallis non-parametric and Dunn's multiple comparison tests.
were signi®cantly different from the control (P # 0:05). The difference in the number of lung tumors between the control group and the SDGsupplemented groups was compared. The median number of lung tumors in the control group was 62, and that in the SDG-treated groups was 38, 36 and 29 (Table 1). The last was signi®cantly different from the control (P # 0:01). The mean number of lung tumors in the control group was 64 ^ 8, and that in groups fed the SDG-supplemented diets was 33, 34 and 48% lower than the control group, respectively. To determine the effect of SDG on the size of metastatic tumors that formed in the lungs, tumor crosssectional area and volume were measured [15,18]. As shown in Table 2, the median cross-sectional area was 0.20 mm 2, and the median volume was 0.09 mm 3 in mice fed the basal diet. Supplementation of SDG to the diets reduced these variables in a dose-dependent manner compared with the controls. The difference between the control group and the groups with SDG at 147 and 293 mmol/kg was signi®cant (P # 0:01). Dietary supplementation with SDG also decreased the mean values of tumor cross-sectional area and volume in a dose-dependent manner. 4. Discussion The results of the present study demonstrate that dietary supplementation with SDG at concentrations equivalent to that provided in the ¯axseed diets [15] reduced the number of lung tumors and tumor crosssectional area and volume compared with the controls.
The dietary content of SDG was positively correlated with both urinary concentration of lignans and the magnitude of the inhibition on metastasis. These results indicate that SDG or mammalian lignans derived from SDG effectively reduced pulmonary metastasis of melanoma cells in mice and retarded the growth of metastatic tumors that developed in the lungs. This supports our previous ®nding that dietary supplementation with ¯axseed reduces experimental metastasis [15], and identi®es SDG as an active antimetastatic constituent of ¯axseed. Results from this study provide the ®rst evidence that dietary lignans reduce experimental metastasis. To metastasize, a malignant cell or a group of cells leave the primary tumor, invade the local tissue, intravasate into the blood circulatory system, arrest in a vascular bed of the target organ, extravasate into the interstitium of the organ, and proliferate to form metastatic tumors. The process can take place once again to yield other metastases. Interventions that block any of these steps can theoretically prevent the spread of malignant cells. In the intravenous injection model used in the present study, the melanoma cells were directly injected into the cardiovascular system. This model eliminates intravasation, but measures the ability of malignant cells to extravasate into the lung tissue. We found that dietary supplementation with SDG decreased the number of metastatic tumors that developed in the lungs. This suggests that SDG or mammalian lignans activated from SDG affect extravasation of malignant cells. Extravasation, as well as intravasation, is a threestep invasive process. It includes the adhesion of
D. Li et al. / Cancer Letters 142 (1999) 91±96
cancer cells to the extracellular matrix, proteolytic degradation, and migration of malignant cells through the degraded region. At present, there have been no publications on lignans and cancer invasion. However, results of in vitro studies show that iso¯avones, another major group of dietary phytoestrogens, affect the invasive behavior of neoplastic cells. This includes the inhibition of cell adhesion to the extracellular matrix proteins [20] and invasion of cancer cells through a reconstituted extracellular matrix barrier [21]. Although exposure of cancer cells to iso¯avones in vitro is not comparable with providing animals iso¯avone-supplemented diets, these results indicate that dietary phytoestrogens may reduce metastasis by inhibiting invasion. Because of the structural and biological similarities of lignans to iso¯avones, the effect of lignans on cancer invasion deserves further investigation. In the present study, we found that tumor crosssectional area and volume of mice fed the SDG diets were decreased in a dose-dependent manner compared with those of mice fed the basal diet. A decrease in tumor size could be due to prolonged retention of tumor cells in the circulatory system or an inhibition of malignant cell proliferation after they take up residence in the lungs. Quantitative analysis on experimental metastasis of B16 melanoma cells has shown that most circulating tumor cells die following injection [19,22]. Approximately 1% of the cells survives for 24 h, and one tenth of them form lung colonies. Therefore, it seems unlikely that retention in the circulation is responsible for the difference in tumor size between the SDG-supplemented groups and the control group. It is more likely that this difference is due to the inhibition of mitosis of malignant cells in the lungs. In vitro experiments show that mammalian lignans inhibit proliferation of tumor cells [12,13]. Flaxseed and SDG inhibit the growth of chemically induced mammary tumors when they are provided to rats after the establishment of the primary tumor [7]. Our results are in agreement with the previous publication that dietary lignans inhibit the growth of the primary tumors [7]. We suggest that lignans also inhibit the growth of the metastatic tumors. Early epidemiologic investigations demonstrate that Japanese breast cancer patients have a higher survival rate and a better prognosis than their Western
95
counterparts [23,24]. Japanese patients are less likely to have histologic evidence of local invasion and regional metastasis than American patients at the time of diagnosis or surgery [25,26]. Although these studies were not designed to correlate the nutritional status of patients with the disease risk, it is unlikely that the differences can be solely explained by ethnicity. Rather, environmental differences between the two populations, particularly in the diet, may be important contributors. A previous clinical trial showed that dietary supplementation with a combination of nutritional antioxidants, in addition to surgical and other therapeutic treatments, prevents further metastasis in breast cancer patients with tumor spread to lymph nodes in axilla [27]. Taken together, these results suggest that dietary habits and nutritional intervention affect the prognosis of post-surgery cancer patients. We have shown that dietary supplementation with soybean [28] and ¯axseed [15] reduce experimental metastasis. A similar inhibitory effect on experimental metastasis was observed when iso¯avones genistein and daidzein were supplemented to the animal diets [29]. The present study demonstrated that dietary supplementation with SDG protects mice against pulmonary metastasis of melanoma cells. Based on these results, we suggest that dietary phytoestrogens can be used as nutritional adjuvants in preventing metastasis in human cancer patients. In summary, the results of the present study demonstrate that dietary supplementation with SDG reduced experimental metastasis of melanoma cells in mice and also inhibited the growth of metastatic tumors that developed in the lungs. We conclude that SDG or mammalian lignans activated from SDG are responsible, at least in part, for the protective effect of dietary ¯axseed on experimental metastasis of melanoma cells in mice. Acknowledgements The authors thank Vivian W. Huang and Mai-Linh Frascarelli, undergraduate students at Creighton University, for participating in this research project, Felicia Cheung, University of Toronto, for technical assistance with the lignan analysis, and Dr. Gleb R. Haynatzki who provided help with statistical analysis
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