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Carcinogenesis Advance Access originally published online on April 5, 2006
Carcinogenesis 2006 27(9):1739-1747; doi:10.1093/carcin/bgl032
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© The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Enhancement of mammary carcinogenesis in two rodent models by silymarin dietary supplements

Barbara Malewicz, Zaisen Wang1, Cheng Jiang, Junming Guo, Margot P. Cleary, Joseph P. Grande2 and Junxuan Lü*

The Hormel Institute, University of Minnesota 801 16th Avenue NE, Austin, MN 55912, USA
1 Agricultural Research and Extension Programs, Langston University PO Box 1730, Langston, OK 73050, USA
2 Department of Laboratory Medicine and Pathology, Mayo Clinic Rochester, MN, USA

*To whom correspondence should be addressed. Tel: +1 507 437 9680; Fax: +1 507 437 9606; Email: jlu{at}hi.umn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Silymarin is a mixture of polyphenolic flavonoids isolated from milk thistle (Silybum marianum) with anticancer activities reported for several organ sites. The present study tested the efficacy of dietary silymarin against mammary carcinogenesis in two rodent models. In the Sprague–Dawley rat model, female rats were fed a purified diet supplemented with none, 0.03, 0.1, 0.3 or 1% (w/w) of silymarin from 21 days of age (DOA) and carcinogenesis was initiated by a single i.p. injection of 1-methyl-1-nitrosourea (MNU) at 51 DOA. Mammary tumor (MT) development was followed till 110 days after carcinogen injection. In the MMTV-neu/HER2 transgenic mouse mammary carcinogenesis model, homozygous transgenic females were fed a purified diet supplemented with none or 0.3% silymarin, either from 28 or 120 DOA and MT development was followed to ~300 DOA. The results showed that dietary silymarin increased the plasma concentration of free and total silibinin, a major component of silymarin, in a dose-dependent manner in the rat, but did not decrease either MT incidence or number. Instead silymarin modestly increased the number of MNU-induced MTs in rats. Similarly, silymarin increased MT incidence and multiplicity and non-MTs in the neu-transgenic mice. In cell culture, treatment of human MCF-7 breast cancer cells with serum-achievable concentrations of silymarin in the rodent models stimulated their growth, in part through an estrogen-like activity. Because silymarin is being used in the treatment of liver cirrhosis and a variety of other human ailments, and is sold as a dietary supplement, our findings add a cautionary note to its application in breast cancer prevention.

Abbreviations: BrdU, bromodeoxyuridine; DOA, days of age; ER, estrogen receptor; MNU, 1-methyl-1-nitrosourea; MT, mammary tumor


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Breast cancer is the most common malignancy of women in the United States with over 200 000 new cases diagnosed every year (1). It is also the second leading cause of cancer mortality in women, next to lung cancer (1). In spite of the sophistication and advances of modern medicine, treatment for metastatic breast cancers is far from satisfactory. Chemoprevention, i.e. the use of either synthetic or naturally occurring agents to inhibit pre-cancerous events, has become recognized as a plausible, cost-effective and necessary approach to reduce cancer morbidity and mortality (2–4). Some classic clinical trials clearly have demonstrated the value of chemoprevention for several human malignancies, notably, a risk reduction of breast cancer by the anti-estrogen tamoxifen (5), and of prostate, colon and lung cancers by selenium (6).

The importance of new agents effective for breast cancer chemoprevention is highlighted by the fact that tamoxifen, the only FDA-approved chemopreventive agent for breast cancer, causes severe adverse effects including an increase of the risk of endometrial cancer (7). Phytochemicals present in fruits, vegetables, tea, wine and some medicinal herbs, especially botanical polyphenolic antioxidants, have in the last decade received increasing attention as potential chemopreventive agents for different human cancers (8). Silymarin, isolated from milk thistle [Silybum marianum (L.) Gaertn], is a mixture of polyphenolic flavonoids with strong antioxidant activity (9–12). Several studies have shown that silymarin protects against hepatotoxicity in rodents (10,11). For several decades silymarin has been used clinically in Europe and Asia for the treatment of liver cirrhosis (13). Recently, it has become popular in the United States as a sole dietary supplement or as a component of various antioxidant mixtures. Studies using different modes of administration to laboratory rodents, rabbits and dogs indicated that silymarin is non-toxic in acute, short- and long-term tests (10,11). Therefore, silymarin could be well suited for continual use as a cancer chemopreventive agent.

Preclinical studies have indicated a potent preventive efficacy of silymarin, regardless of its administration routes, for mouse skin cancer induced either by chemical carcinogens or UV irradiation (14–19). In addition, recent animal studies have shown that silymarin inhibits chemically-induced carcinogenesis of the colon (20), tongue (21) and bladder (22). An increasing body of literature suggests that silymarin may prevent the initiation and/or development of breast cancer. Silymarin was shown to inhibit 7,12-dimethylbenz[a]anthracene-initiated and 12-O-tetradecanoylphorbol-13-acetate promoted formation of mammary lesions in an organoid culture model (23). In cell culture, a direct exposure of breast or other cancer cells to silymarin or its major component silibinin leads to an inhibition of mitogenic signaling molecules, and a selective induction of cyclin-dependent kinase inhibitor Kip1/p27, and ultimately causes cellular G1 arrest, followed by cell growth inhibition, differentiation and/or apoptosis (24–27). Moreover, we have shown that silymarin expresses strong anti-angiogenic activity in cell culture (28). Based on these findings, we hypothesized that silymarin could exert a chemopreventive activity against mammary carcinogenesis in vivo.

We sought to test this hypothesis using two rodent models as follows: 1-methyl-1-nitrosourea (MNU)-induced mammary carcinogenesis in Sprague–Dawley rats; and MMTV-neu-transgenic mice bearing a human breast cancer proto-oncogene Her2/neu. Owing to the unexpected finding of an enhancing effect of silymarin on mammary carcinogenesis in both rodent models, we also carried out cell culture studies with the estrogen-dependent MCF-7 human breast cancer cells to explore potential mechanisms underlying the risk enhancement.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Sprague–Dawley rat model study
Diet
Silymarin was purchased from Sigma-Aldrich (St Louis, MO) as an extra fine powder. Silymarin was mixed in AIN-93G semipurified diet at 0.03, 0.1, 0.3 and 1% (w/w). The AIN-93G formulation (29) was used to minimize potential dietary interactions between silymarin and other nutritive and non-nutritive ingredients of a rodent chow, which is based mainly on natural feed stuff. Diets (in mesh form) were made every 3 weeks and stored at –20°C in the dark to minimize photo destruction of silymarin. Fresh diets were provided twice a week at the time of cage change.

Animals and dietary regiments
Female Sprague–Dawley rats were obtained at 21 days of age (DOA) from Taconic Farms. Groups of 30 rats were fed from 21 DOA either the basal diet or the diet supplemented with varying levels of silymarin. Body weights were recorded twice a week for the first 2 months, and once weekly thereafter. The animals were housed in an AAALAC-accredited animal facility with 12-h light/dark cycle. The study was approved by the Institutional Animal Care and Use Committee (IACUC) of AMC Cancer Research Center (Lakewood, CO) and was carried out at that institution while the senior author (J.L.) was on its staff.

Mammary carcinogenesis
At 51 DOA, each animal was given a single i.p. injection of MNU (50 mg/kg body wt) to initiate mammary carcinogenesis (30). Starting at 28 days after MNU injection, the rats were palpated twice weekly for mammary tumors (MTs). When a tumor was palpated, its location and date of detection were recorded. The experiment was terminated at 110 days post-carcinogen injection, when the tumor incidence and multiplicity data did not support a chemopreventive effect of the dosages of silymarin used.

Necropsy
Rats were euthanized by carbon dioxide inhalation and cervical dislocation. Blood was taken from approximately one-third of the animals in each group for plasma preparation. Plasma samples were stored at –80°C until analyzed for silibinin content. Tumors were dissected and trimmed off capsular materials and weighed. A small piece of each MT was fixed in neutral formalin for histological classification after hematoxylin and eosin staining. Only histologically verified adenocarcinomas were included in cancer incidence and multiplicity data analyses.

Chemical analyses
Free silibinin was directly extracted with ethyl acetate from 200 µl plasma sample. For total silibinin, 100 µl plasma sample was first digested with ß-glucuronidase and sulfatase, and then extracted with ethyl acetate. Silibinin content was quantified by HPLC analysis as described in detail recently (31) and was carried out at the Pharmacology Core Facility of University of Colorado School of Pharmacy, Denver, CO.

Transgenic mouse model study
Experimental animals
MMTV-neu-transgenic mice (wild-type) new is a well-established model (32) to study human breast cancer linked to an overexpression of Her2/ERB2 proto-oncogene (33,34). Experimental animals were obtained by mating homozygous transgenic males and females from line 202 (32). Semiquantitative multiplex PCR was performed to confirm the homozygosity of the neu-transgene in the experimental mice using a modification of a reported procedure (35). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was used as normalization control for PCR analysis.

Diet
The basal control diet consisted of a modified AIN-93M diet with increased fat contents in order to shorten the latency of MTs (35). For that purpose AIN-93M diet was supplemented with soybean oil at the amount of 154.5 g/kg, which translated to 33.5% of calorie intake from fat, approximating the average fat intake of Americans. Silymarin was added to the basal diet at 0.3% w/w. Diets were prepared by Harlan Teklad (Madison, WI) into pellets every 6 months and were kept at –20°C to minimize their oxidization.

Mammary carcinogenesis experiment
Mice were assigned at 28 DOA to one of three groups. Care was taken to equally assign siblings into each group. The first (control) group of animals was fed the basal diet throughout the study. The second (early silymarin) group of mice was fed silymarin-supplemented diet throughout the study. The third (late silymarin) group was first fed the basal diet until 120 DOA, then was fed the silymarin-supplemented diet throughout the rest of the experiment. Four mice were housed per cage. However, along the duration of the experiment some animals were separated owing to fighting. Food and water was given ad libitum. The animal room was maintained at 22°C on a 12-h light/dark cycle and 50% humidity. The mice were observed daily for any changes in overall health and behavior, or for any sign of distress. The mice were weighed every other week to monitor their growth. Starting from 120 DOA, mice were palpated weekly to detect MTs. Mice were euthanized when their tumors were ulcerated or reached the size of ~20 mm in diameter, or when they experienced excessive distress as indicated by loss of body weight >20%, or severely injured. The study was terminated after ~10 months, when the incidence of MTs in the control group reached ~50%. The study was approved by the IACUC of the University of Minnesota, and carried out at the Hormel Institute's AAALAC-accredited animal facility.

Necropsy and tissue processing
Mice were euthanized by carbon dioxide inhalation. During necropsy MTs and other tumors or organ/tissue abnormalities were identified and collected for histopathological verification. MTs were dissected, trimmed off capsular materials and weighed. A small piece of each MT and other abnormalities was fixed in 10% neutral formalin, and stained with hematoxylin and eosin. Histopathology was examined by an experienced clinical pathologist (J.P.G.) on a ‘blind’ basis to avoid any bias.

MCF-7 cell culture study
Human MCF-7 breast cancer cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) and maintained in EMEM medium (ATCC) supplemented with 10 µg/ml insulin and 10% fetal bovine serum (FBS) (Atlanta Biologicals, Lawrenceville, GA). To assess the estrogen receptor (ER) agonist activity, we seeded MCF-7 cells in phenol red-free IMEM medium (Bioscience, Rockville, MD) containing 5% charcoal-stripped FBS (CS-FBS, Atlanta Biologicals) in 6-well plates for 2 days to decrease background ER signaling and treated the cells with increasing levels of silymarin in the absence or presence of 17-ß-estradiol (E2, Sigma) for 7 days. The cells were fixed and stained with crystal violet and photographed. The cell number was estimated by the optical density of the dissolved crystal violet dye in 70% ethanol. The pure anti-estrogen compound Faslodex (ICI 182,780, Tocris Bioscience, Avonmouth, UK) was used in some experiments to establish the specificity of the estrogen-like activity of silymarin.

Cell proliferation rate was estimated by bromodeoxyuridine (BrdU, Sigma) incorporation into S-phase cells. In brief, after 2 days of exposure to silymarin, BrdU (18 µg/ml) was added to the cell culture medium for 2 h and the cells were harvested by trypsinization and processed for BrdU immunofluorescence. The cellular DNA was also stained with propidium iodide. BrdU and cellular DNA contents were detected by two color flow cytometry. Immunoblot analyses of the expression of selected proteins associated with mitogenesis signaling and cell cycle regulation were carried out to determine probably molecular mediators/targets for enhancing cell proliferation by silymarin.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
The effects of silymarin on MNU-induced MTs in Sprague–Dawley rats
The effect of silymarin feeding on body weight
Before we initiated the current mammary cancer prevention project, all published anticarcinogenesis studies had used topical application of silymarin directly on the mouse skin (15–19). Because there was no information available on the effect of this agent delivered to Sprague–Dawley rats via the dietary route, a pilot experiment was performed on four groups of rats to provide such information. Four rats at 8 weeks of age were assigned into each of the dietary groups and were fed for 2 weeks the AIN-93G diet supplemented with 0, 0.01, 0.1 and 1% of silymarin. The body weight gain was (mean ± SD) 10.1 ± 4.3, 9.5 ± 1.7, 13.8 ± 2.5 and 16.0 ± 3.0 g per rat, respectively, indicating that a short-term silymarin consumption did not inhibit growth of the rats. Based on these results we chose 1% as the highest dose to carry out the carcinogenesis/prevention experiment.

Silymarin intake over the duration of the carcinogenesis study of 140 days had minimal adverse effects on body weight (Figure 1A). At the initial growth stage, i.e. before MNU injection, there was no difference in the average body weights of rats from all dietary groups. After MNU injection, there was a small decrease of the growth rate for rats fed the silymarin-supplemented diets. However, there was no obvious dose–response relationship between silymarin level and the body weight retardation, which at the end of experiment was only 3.2–7.4% of control level. These results further supported the premise of well-tolerated nature of this agent for long-term oral use.


Figure 1
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  		Fig. 1 (A)The Effect of Silymarin on body weight of female Sprague–Dawley rats. Data represent the mean of 30 (initial number) rats/group that were fed silymarin-containing diets from 21 DOA (indicated by arrowhead) and were each given a single i.p. injection of 25 mg MNU/kg body wt on 51 DOA (denoted by long arrow). The experiment was terminated on 110 days post-MNU injection. (B) Plasma silibinin content of rats at termination of experiment (upper plot, free; lower plot, free plus conjugated). Data represent the mean of 8–10 samples/group. Vertical bar denotes 1 SEM.

 
Plasma silibinin
The plasma levels of free and total, i.e. free plus conjugated, silibinin (Figure 1B), the major active component of silymarin, at the termination of the experiment were increased in a concentration-dependent manner. The free and total silibinin reached ~5 and 35 µg/ml, respectively, by the highest dietary dose.

The effect of silymarin supplement on MTs in Sprague–Dawley rats
Figure 2A shows the incidence of MTs, i.e. percentage of rats with at least one MT, as a function of post-carcinogen exposure time. Dietary silymarin neither delayed the time it took to reach 50% incidence of MTs nor decreased the number of tumor-bearing animals at the end of the experiment, even though there appeared to be an initial delay of tumor occurrence in the group fed 0.1% silymarin. Numerically, the number of tumor-bearing rats at the end of the experiment was greater in the silymarin-fed groups than in the control group. Yet, there was no clear dose-dependency on the silymarin content in the diet (Figure 2A).


Figure 2
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  		Fig. 2 Effects of dietary silymarin on (A) the proportion of rats bearing at least one MT (tumor incidence) and (B) the mean number of palpable mammary carcinomas per rat (tumor multiplicity) as a function of days post-MNU injection. Starting 28 days after carcinogen administration, rats were palpated for MTs. MTs that had been confirmed histologically as adenocarcinomas were used for the plots. (C) The effect of dietary silymarin on the number of mammary adenocarcinomas per carcinogen-exposed rat at final necropsy. Data are mean ± SEM, n = 30 rat/group.

 
Similarly, the number of palpable MTs developed per carcinogen-exposed rat (Figure 2B) was not decreased by silymarin consumption. On the contrary, the rats fed silymarin-containing diets developed ~30–40% more tumors than animals in the control group by the end of the study (Figure 2C). There was no apparent dose–response effect of silymarin.

Owing to heavy tumor burden, 3, 5, 3, 5 and 7 rats in the control and 0.03, 0.1, 0.3 and 1% of silymarin groups, respectively, had to be terminated before the end of the experiment. The tumor burden (sum of weight of tumors per tumor-bearing animal) determined at the end of the study for the remaining rats did not differ significantly between the groups (Table I). Taken together, these data suggest that there was no protective effect of silymarin on tumor initiation and growth in Sprague–Dawley rats. Instead, silymarin exerted a modest and statistically significant enhancement effect in this model.


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  		Table I The effect of dietary silymarin on MNU-induced mammary carcinogenesis in female Sprague–Dawley rats

 
The effects of silymarin on MTs in MMTV-neu-transgenic mice
The unexpected results obtained with the MNU-induced mammary carcinogenesis model raised the question whether the enhancing effect is limited to chemically-induced carcinogenesis only or if this effect is of a more general nature. Therefore, we examined the effect of silymarin on a different model of mammary carcinogenesis in the MMTV-neu-transgenic mice. All female offspring from our in-house breeding colony were genotyped by PCR to ensure the homozygous status for the neu-transgene. Figure 3A shows an example of the detection method capable of distinguishing heterozygous (lane 1) from homozygous mice (lanes 2–6 and 8) and non-transgenic mice (lane 7).


Figure 3
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  		Fig. 3 (A)Representative agarose gel electrophoresis results of the multiplex PCR genotyping analysis of tail DNA from female MMTV-neu mice. Lane 1, DNA from a heterozygous MMTV-neu mouse (+/–); lanes 2–6, DNA samples from experimental mice; lane 7, DNA from a non-transgenic mouse (–/–); lane 8, DNA from a homozygous MMTV-neu mouse (+/+). (B) The effects of silymarin on body weight of female MMTV-neu mice. Data represent the mean obtained for 55 or 56 (initial number) mice/group that were fed control diet or 0.3% silymarin containing diet either from 28 DOA (the early silymarin group) or from 120 DOA (the late silymarin group). (C) The effect of silymarin on the onset of MTs in MMTV-neu mice. Symbols for the groups are the same as in B. The effective total number for mice for the control group was 50 mice, for early silymarin group was 53 mice, for late silymarin group was 51 mice. For the final tumor incidence, {chi}2 = 3.32 for the combined silymarin groups versus control group, 0.05 < P < 0.1.

 
Based on the results obtained from the rat model, we chose only the 0.3% silymarin diet for the mouse experiment. In addition, we wanted to address the question whether the timing of dietary supplementation in relation to mammary gland maturity and tumor lesion development modulate the effect of silymarin on mammary carcinogenesis. Therefore, one group of mice was fed the silymarin-supplemented diet starting at 120 DOA (late silymarin group), after their sexual maturation was well-established and early stage mammary lesions would have developed (32).

The effect of silymarin on mouse body weight
The average body weight at the beginning of the study (28 DOA) was 17.6 (± 1.7 SD), 17.5 (± 1.7) and 17.6 (± 1.6) g for control, early silymarin and late silymarin group, respectively. Initially, 55 or 56 mice were assigned to each dietary group, and then these numbers declined during the progress of the experiment owing to attrition as well as tumor burden. The data on mouse body weight shown in Figure 3B represent the mean obtained on all mice alive at the time of analysis. Silymarin supplement did not alter the growth of mice. In addition, there were no apparent differences among the experimental groups in the appearance or behavior of the mice. These mice were quite active, and any lethargy could have been easily observed. As expected, the results indicated that silymarin dietary supplement given as early as 4 weeks of age did not hamper the mouse growth.

The effect of silymarin on mammary carcinogenesis in MMTV-neu-transgenic mice
The transgenic mice became quite aggressive as they matured. Despite best attempts to separate them, some of the mice were found dead or were badly injured and had to be euthanized. Consequently, there was ~5–10% loss of mice owing to attrition. Because most of these occurred before the onset of MTs, these mice were removed from the study without influencing the conclusions drawn regarding the effects of silymarin.

As shown in Figure 3C, the onset of MTs started at the age of 29 weeks in the late silymarin group, and 32 weeks in the control as well as in the early silymarin group. Half of the mice in the early silymarin and the late silymarin groups developed MTs at 38 and 37 weeks of age, respectively. However, in the control group it took 45 weeks to reach 48% incidence. Feeding from 28 DOA (early silymarin group, immature mammary glands) increased the numbers of mice with MT (incidence) toward the end of the experiment, but did not speed up the initial onset of MT.

Numerically, the MT incidence at the end of experiment was higher by 30 and 35% in the early and late silymarin groups, respectively, as compared with the control group (Table II). The number of MTs from the early silymarin and late silymarin groups were 40 and 39%, respectively, higher than in the control group. This increase was mainly owing to an increase in the number of mice bearing multiple MTs regardless of the length of silymarin supplementation (Table II). The tumor burden (sum of all tumor weight) per tumor-bearing mouse was similar among the three groups.


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  		Table II Effect of silymarin-supplemented diet on MTs in female MMTV-neu-transgenic mice

 
Non-mammary abnormalities detected in MMTV-neu-transgenic mice
The transgenic mice developed additional abnormalities (Table III). It is noteworthy that seven out of the eight non-mammary abnormalities occurred in mice fed the silymarin-supplemented diet. Out of 154 mice in the study, six mice developed tumors in their ovaries. Four of the mice (one in control group and three in late silymarin group) had tumors with histological features consistent with teratoma (multiple cystic structures containing squamous epithelium and keratin, foci of cartilage and areas containing primitive epithelium). In the early silymarin group one mouse developed a cystic ovarian neoplasm consisting primarily of keratin-filled cysts, bone and epithelial-lined cystic tissue, and one mouse developed a high-grade ovarian adenocarcinoma. In the late silymarin group there was one case of salivary gland adenocarcinoma, and one mouse developed a high-grade lymphoma/poorly-differentiated carcinoma that spread over the fallopian tubes, spleen, pancreas and liver.


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  		Table III Non-MTs in female MMTV-neu-transgenic mice

 
Effects of silymarin on growth of MCF-7 cells in cell culture
The results from both rodent models prompted us to investigate the possible mechanisms for silymarin to enhance breast cancer growth. Since it has been suggested that silymarin may have phytoestrogen activities (36), we focused on the possibility of serum-achievable levels of silymarin might act as an ER agonist in the estrogen-responsive human MCF-7 breast cancer cells. As expected, the addition of 17-ß-estradiol (E2) significantly increased cell growth (Figure 4A, +0.1 nM E2 control versus –E2 control). Increasing E2 level stimulated cell growth in a curve–linear manner, indicating that 0.1 nM E2 represented a submaximal stimulation because 1 nM E2 promoted an even greater cell accumulation (Figure 4B). In the absence and presence of the submaximal E2, silymarin in the range of 5–20 µg/ml increased MCF-7 growth in a concentration-dependent manner (Figure 4A and C) and the two agents produced an additive growth stimulation (Figure 4C). However, at the exposure concentration of 50 µg/ml and higher, silymarin treatment decreased cell number in a concentration-dependent manner (Figure 4A and C), regardless of E2 level.


Figure 4
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  		Fig. 4 (A)Typical data obtained in 6-well plates showing the growth of MCF-7 cells as a function of increasing concentrations of silymarin in the absence (–E2) and presence (+E2) of 0.1 nM 17-ß-estradiol. Cells were seeded in phenol red-free cell culture medium with 5% charcoal-stripped serum for 2 days and treated with increasing levels of silymarin as indicated and cultured for 7 additional days before fixation and staining with crystal violet. (B) Effect of increasing E2 on cell number estimated by the optical density of dissolved crystal violet in 70% ethanol. (C) Quantification of the biphasic growth response of MCF-7 cells as a function of increasing concentrations of silymarin in the absence and presence of +E2. Data are based on three independent experiments, error bars indicate SD. ANOVA and t-tests show statistically (P < 0.05) significant increases of cell number within the concentration range of 5–20 µg/ml with and without supplemental estrogen. (D) The inhibitory effect of an anti-estrogen Faslodex on silymarin or E2-stimulated growth of MCF-7 cells.

 
To further confirm the critical role of estrogen-ER signaling, we co-treated MCF-7 cells with silymarin and the anti-estrogen Faslodex. As shown in Figure 4D, Faslodex decreased cell growth in a concentration-dependent manner in the absence of added E2 (columns 2 and 3 versus 1). It effectively blocked silymarin-induced (columns 5 and 6 versus 4) as well as E2-induced cell growth (column 8 versus 7).

Next, we investigated the effect of silymarin on the rate of cell proliferation using BrdU incorporation as a marker (Figure 5A). After the MCF-7 cells had been treated with silymarin for 2 days in the absence of added E2, BrdU was added for 2 h to pulse-label cells actively engaged in DNA replication. As shown in Figure 5B, within the serum-achievable concentration range, silymarin induced a near-linear stimulatory effect on BrdU-positive cells.


Figure 5
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  		Fig. 5 (A)Flow cytometric detection of BrdU-labeled MCF-7 cells engaged in DNA replication during S-phase. Cells were seeded and treated as in Figure 4 without added E2. After 2 days of silymarin treatment, the cells were incubated with BrdU for 2 h before being harvested for immunofluorescence detection. Cells not incubated with BrdU (–BrdU panel) were used as negative control to define the positive BrdU signals. (B) Effects of increasing silymarin on the BrdU index. The results represent two independent experiments. (C) Immunoblot analyses of the expression of selected biochemical markers of cell cycle regulation in MCF-7 cells identically treated as in B, without BrdU. The plot shows c-Myc and cyclin E protein abundance by densitometric analyses.

 
Immunoblot analyses of MCF-7 cells identically treated for 2 days with silymarin as above showed a concentration-dependent induction of c-Myc protein (Figure 5C), which is a known early response gene of estrogen stimulation (37,38 and references therein). Consistent with an increased S-phase population, the abundance of the S-phase-specific cyclin E was increased by silymarin, although the extent of change was about half of that observed for c-Myc. The enhanced proliferative effect was observed without a significant effect on the expression level of the ER-{alpha} protein or the early G1-specific cyclin D1. Together, the cell culture results have indicated that serum-achievable levels of silymarin increase ER signaling and cell proliferation.


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The results from the two rodent models of mammary carcinogenesis were quite unexpected. They were in sharp contrast to the results of several preclinical studies in animal models of carcinogenesis in other organ sites. For example, dietary supplement with silymarin has been shown to decrease the tumor incidence and number in chemically-induced rat models of carcinogenesis in the colon (20), tongue (21) and bladder (22) and to decrease the growth of human prostate cancer and lung cancer xenografts in immunodeficient mice (39,40). These reports were consistent with the potent protective action of silymarin observed in the mouse skin carcinogenesis models (15–19). Instead of the anticipated protective effects, we observed a moderate increase of MNU-induced carcinogenesis in Sprague–Dawley rats (Figure 2B and Table I) and oncogene-driven MTs in MMTV-neu mice (Figure 3C and Table II). It should be noted that in the mammary carcinogenesis models, in particular, adverse effect of agents on the health or physiology of the animals as reflected by reduced body weight is usually associated with a reduced tumor outcome (41,42). Chronic silymarin feeding for ~140 days to rats and 300 days to mice showed very little, if any, adverse effects on body weight gains (Figure 1A and Figure 3B), supporting the well-tolerated nature of this agent. Thus, since we have observed negligible effect of silymarin on body weights in both animal models, it is unlikely that its ‘true’ chemopreventive effect might have been compromised owing to body weight impact.

In view of the anticancer activities in the other organ sites, the unexpected enhancement effects in mammary models support a specific enhancement activity of silymarin in this estrogen-sensitive organ. This notion was further supported by cell culture experiments in which we focused on the estrogen-like activity of silymarin in the estrogen-dependent MCF-7 cell culture model (Figures 4 and 5). The plasma silibinin levels achieved in the rats (up to 5 µg free per ml) (Figure 1B) were in the same range as those reported for mice fed a silymarin-supplemented diet that exerted a chemopreventive effect against prostate cancer (31,39). Because silibinin is the major constituent of silymarin, these values would correspond to ~10 µg/ml free silymarin in cell culture medium. The cell colony growth experiments (Figure 4A and C) revealed biphasic response curves as a function of silymarin concentration. Within the serum-achievable concentration range, silymarin and submaximal E2 produced additive stimulation of cell growth in a concentration-dependent manner (Figure 4A and C). Conversely, the growth stimulation by silymarin as well as by E2 was reversed by blocking ER function with Faslodex (Figure 4D). These results support stimulation of ER signaling by silymarin as a major pathway to stimulate breast cancer cell growth, which was clearly reflected by the linear increase of S-phase cells when the silymarin concentration was elevated (Figure 5A). In further support of this notion, silymarin treatment increased the abundance of c-Myc (Figure 5C), which is a known estrogen-responsive gene product (37,38). Because estrogen is an endogenous promoter for breast cancer and for the rodent mammary carcinogenesis models studied here (42–48), these cell culture results provide a plausible mechanism to account for the enhanced mammary carcinogenesis in the rodent models.

The cell culture data also help to reconcile with growth suppression effects reported in earlier literature with human breast cancer cell lines (24,49). We also observed the strong growth inhibitory effect at 50–100 µg/ml silymarin (Figure 4A). However, such levels may only be achievable through pharmacological means and may not be applicable to chemoprevention through dietary supplement.

Because of the experimental nature of the animal models in our study, the data presented here do not preclude a possible benefit of silymarin for breast cancer prevention in women. They nevertheless add a cautionary note to such an application. The finding of an apparent enhancement of non-MTs by silymarin consumption in the neu-transgenic mice, chiefly in the ovary (Table III), further supports this notion. However, the risk implications of consuming silymarin will be more meaningful in the context of currently used doses in humans. For example, silymarin is used as a dietary supplement for a variety of ailments in the range of 200–600 mg/day. Available human pharmacokinetic studies with silymarin or silybin have shown poor bioavailability (50,51). For example, plasma level peaks at 0.05 µg/ml with a single oral dose of 360 mg of silybin, the major component of silymarin. When silybin is complexed with phosphatidylcholine into phytosomes, the plasma peak concentration can be improved to 0.2–0.3 µg/ml (50). In rodent studies, plasma silibinin levels associated with either preventive effect for prostate cancer (39) or enhancement effect in our studies (Figure 1) are 5–10 times higher than in human plasma. Therefore, the risk and benefit assessment of silymarin consumption will require careful consideration of dosage as well as differences in metabolism between rodents and men.

Since silymarin is a natural product of four major silibinin compounds, and several minor components (8–12), each of these components may exert different biological activities as have been demonstrated by a recent study of silibinin derivatives in a prostate cancer cell culture model (52). It will be essential and necessary in future studies to identify the silymarin components that confer the estrogen-like and growth stimulatory activities. Studies of the structure and activity relationship of the different silymarin constituents and silibinin derivatives in terms of their growth stimulatory activities may help to identify compounds that lack the enhancement activity on mammary cancer risk or even be chemopreventive for mammary cancer.

In summary, our data did not support the hypothesis regarding a chemopreventive effect of silymarin dietary supplement, delivered from pre-pubescence on the chemical induction of mammary carcinogenesis in the rats, or delivered from pre- and post-pubescence to mice on their oncogene-driven MTs. On the contrary, consumption of silymarin led to modest increases of MTs in both models. We showed that silymarin within the serum-achievable concentration range in the rodents stimulated MCF-7 breast cancer cell growth in cell culture, probably through estrogen-like activities. Given the positive preventive activities in animal models of other organ sites (15–22,39), we believe that additional studies are warranted to define the structural features of silymarin compounds that confer the estrogen-like activity and to develop derivatives that eliminate this activity. Before such information is attained, a cautionary note is prudent for women who are on, or intend to use, silymarin-containing dietary supplements for breast cancer prevention purposes.


   Acknowledgments
 
We thank Melissa Bonorden for advices on genotyping of neu-transgenic mice, Todd Schuster for assistance with flow cytometry detection of BrdU index and Andria Hansen for editorial support. This work was supported in part by grant BCTR-2000-297 from the Susan G. Komen Breast Cancer Foundation, the Hormel Foundation and the Eagle's Telethon (J.L.), and by grant CA101858 from the National Cancer Institute (M.P.C.). The rat study was carried out when Z.W. and J.L. were on the staff of the AMC Cancer Research Center, Denver, CO.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Jemal A., Murray T., Ward E., Samuels A., Tiwari R.C., Ghafoor A., Feuer E.J., Thun M.J. (2005) Cancer statistics, 2005. CA Cancer J. Clin. 55:10–30.[Abstract/Free Full Text]
  2. Sporn M.B. and Suh N. (2002) Chemoprevention: an essential approach to controlling cancer. Nat. Rev. Cancer 2:537–543.[CrossRef][ISI][Medline]
  3. Hong W.K. and Sporn M.B. (1997) Recent advances in chemoprevention of cancer. Science 278:1073–1077.[Abstract/Free Full Text]
  4. Wattenberg L.W. (1997) An overview of chemoprevention: current status and future prospects. Proc. Soc. Exp. Biol. Med. 216:133–141.[Abstract]
  5. Fisher B., Costantino J.P., Wickerham D.L., et al. (1998) Tamoxifen for prevention of breast cancer: report of the National Surgical Adjuvant Breast and Bowel Project P-1 Study. J. Natl Cancer Inst. 90:1371–1388.[Abstract/Free Full Text]
  6. Clark L.C., Combs G.F. Jr, Turnbull B.W., et al. (1996) Effects of selenium supplementation for cancer prevention in patients with carcinoma of the skin. A randomized controlled trial. Nutritional Prevention of Cancer Study Group. JAMA 276:1957–1963.[Abstract]
  7. Brown K. (2002) Breast cancer chemoprevention: risk-benefit effects of the antioestrogen tamoxifen. Expert Opin. Drug Saf. 1:253–267.[CrossRef][Medline]
  8. Dragsted L.O. (1998) Natural antioxidants in chemoprevention. Arch. Toxicol. Suppl. 20:209–226.[Medline]
  9. Wagner H., Horhammer L., Munster R. (1968) On the chemistry of silymarin (silybin), the active principle of the fruits from Silybum marianum (L.) Gaertn. (Carduus marianus L.). Arzneimittelforschung 18:688–696.[Medline]
  10. Hahn G., Lehmann H.D., Kurten M., Uebel H., Vogel G. (1968) [On the pharmacology and toxicology of silymarin, an antihepatotoxic active principle from Silybum marianum (L.) Gaertn]. Arzneimittelforschung 18:698–704.[Medline]
  11. Vogel G., Trost W., Braatz R., Odenthal K.P., Brusewitz G., Antweiler H., Seeger R. (1975) Pharmacodynamics, site and mechanism of action of silymarin, the antihepatoxic principle from Silybum mar. (L) Gaertn. 1. Acute toxicology or tolerance, general and specific (liver-) pharmacology. Arzneimittelforschung 25:82–89.[Medline]
  12. Valenzuela A., Guerra R., Videla L.A. (1986) Antioxidant properties of the flavonoids silybin and (+)-cyanidanol-3: comparison with butylated hydroxyanisole and butylated hydroxytoluene. Planta. Med. 438–440.
  13. Ferenci P., Dragosics B., Dittrich H., Frank H., Benda L., Lochs H., Meryn S., Base W., Schneider B. (1989) Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver. J. Hepatol. 9:105–113.[CrossRef][ISI][Medline]
  14. Singh R.P. and Agarwal R. (2002) Flavonoid antioxidant silymarin and skin cancer. Antioxid Redox Signal 4:655–663.[CrossRef][ISI][Medline]
  15. Agarwal R., Katiyar S.K., Lundgren D.W., Mukhtar H. (1994) Inhibitory effect of silymarin, an anti-hepatotoxic flavonoid, on 12-O-tetradecanoylphorbol-13-acetate-induced epidermal ornithine decarboxylase activity and mRNA in SENCAR mice. Carcinogenesis 15:1099–1103.[Abstract/Free Full Text]
  16. Katiyar S.K., Korman N.J., Mukhtar H., Agarwal R. (1997) Protective effects of silymarin against photocarcinogenesis in a mouse skin model. J. Natl Cancer Inst. 89:556–566.[Abstract/Free Full Text]
  17. Zi X., Mukhtar H., Agarwal R. (1997) Novel cancer chemopreventive effects of a flavonoid antioxidant silymarin: inhibition of mRNA expression of an endogenous tumor promoter TNF alpha. Biochem. Biophys. Res. Commun. 239:334–339.[CrossRef][ISI][Medline]
  18. Ahmad N., Gali H., Javed S., Agarwal R. (1998) Skin cancer chemopreventive effects of a flavonoid antioxidant silymarin are mediated via impairment of receptor tyrosine kinase signaling and perturbation in cell cycle progression. Biochem. Biophys. Res. Commun. 247:294–301.[CrossRef][ISI][Medline]
  19. Lahiri-Chatterjee M., Katiyar S.K., Mohan R.R., Agarwal R. (1999) A flavonoid antioxidant, silymarin, affords exceptionally high protection against tumor promotion in the SENCAR mouse skin tumorigenesis model. Cancer Res. 59:622–632.[Abstract/Free Full Text]
  20. Kohno H., Tanaka T., Kawabata K., Hirose Y., Sugie S., Tsuda H., Mori H. (2002) Silymarin, a naturally occurring polyphenolic antioxidant flavonoid, inhibits azoxymethane-induced colon carcinogenesis in male F344 rats. Int. J. Cancer 101:461–468.[CrossRef][ISI][Medline]
  21. Yanaida Y., Kohno H., Yoshida K., Hirose Y., Yamada Y., Mori H., Tanaka T. (2002) Dietary silymarin suppresses 4-nitroquinoline 1-oxide-induced tongue carcinogenesis in male F344 rats. Carcinogenesis 23:787–794.[Abstract/Free Full Text]
  22. Vinh P.Q., Sugie S., Tanaka T., Hara A., Yamada Y., Katayama M., Deguchi T., Mori H. (2002) Chemopreventive effects of a flavonoid antioxidant silymarin on N-butyl-N-(4-hydroxybutyl)nitrosamine-induced urinary bladder carcinogenesis in male ICR mice. Jpn. J. Cancer Res. 93:42–49.[CrossRef][ISI]
  23. Mehta R.G. and Moon R.C. (1991) Characterization of effective chemopreventive agents in mammary gland in vitro using an initiation-promotion protocol. Anticancer Res. 11:593–596.[ISI][Medline]
  24. Zi X., Feyes D.K., Agarwal R. (1998) Anticarcinogenic effect of a flavonoid antioxidant, silymarin, in human breast cancer cells MDA-MB 468: induction of G1 arrest through an increase in Cip1/p21 concomitant with a decrease in kinase activity of cyclin-dependent kinases and associated cyclins. Clin. Cancer Res. 4:1055–1064.[Abstract]
  25. Zi X., Grasso A.W., Kung H.J., Agarwal R. (1998) A flavonoid antioxidant, silymarin, inhibits activation of erbB1 signaling and induces cyclin-dependent kinase inhibitors, G1 arrest, and anticarcinogenic effects in human prostate carcinoma DU145 cells. Cancer Res. 58:1920–1929.[Abstract/Free Full Text]
  26. Zi X. and Agarwal R. (1999) Silibinin decreases prostate-specific antigen with cell growth inhibition via G1 arrest, leading to differentiation of prostate carcinoma cells: implications for prostate cancer intervention. Proc. Natl Acad. Sci. USA 96:7490–7495.[Abstract/Free Full Text]
  27. Agarwal R. (2000) Cell signaling and regulators of cell cycle as molecular targets for prostate cancer prevention by dietary agents. Biochem. Pharmacol. 60:1051–1059.[CrossRef][ISI][Medline]
  28. Jiang C., Agarwal R., Lu J. (2000) Anti-angiogenic potential of a cancer chemopreventive flavonoid antioxidant, silymarin: inhibition of key attributes of vascular endothelial cells and angiogenic cytokine secretion by cancer epithelial cells. Biochem. Biophys. Res. Commun. 276:371–378.[CrossRef][ISI][Medline]
  29. Reeves P.G., Nielsen F.H., Fahey G.C. Jr. (1993) AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 123:1939–1951.[Abstract/Free Full Text]
  30. Thompson H.J. and Adlakha H. (1991) Dose-responsive induction of mammary gland carcinomas by the intraperitoneal injection of 1-methyl-1-nitrosourea. Cancer Res. 51:3411–3415.[Abstract/Free Full Text]
  31. Zhao J. and Agarwal R. (1999) Tissue distribution of silibinin, the major active constituent of silymarin, in mice and its association with enhancement of phase II enzymes: implications in cancer chemoprevention. Carcinogenesis 20:2101–2108.[Abstract/Free Full Text]
  32. Guy C.T., Webster M.A., Schaller M., Parsons T.J., Cardiff R.D., Muller W.J. (1992) Expression of the neu protooncogene in the mammary epithelium of transgenic mice induces metastatic disease. Proc. Natl Acad. Sci. USA 89:10578–10582.[Abstract/Free Full Text]
  33. Slamon D.J., Clark G.M., Wong S.G., Levin W.J., Ullrich A., McGuire W.L. (1987) Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 235:177–182.[Abstract/Free Full Text]
  34. Slamon D.J., Godolphin W., Jones L.A., et al. (1989) Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244:707–712.[Abstract/Free Full Text]
  35. Pape-Ansorge K.A., Grande J.P., Christensen T.A., Maihle N.J., Cleary M.P. (2002) Effect of moderate caloric restriction and/or weight cycling on mammary tumor incidence and latency in MMTV-Neu female mice. Nutr. Cancer 44:162–168.[CrossRef][Medline]
  36. Scambia G., De Vincenzo R., Ranelletti F.O., Panici P.B., Ferrandina G., D'Agostino G., Fattorossi A., Bombardelli E., Mancuso S. (1996) Antiproliferative effect of silybin on gynaecological malignancies: synergism with cisplatin and doxorubicin. Eur. J. Cancer 32A:877–882.[CrossRef]
  37. Watson P.H., Pon R.T., Shiu R.P. (1991) Inhibition of c-myc expression by phosphorothioate antisense oligonucleotide identifies a critical role for c-myc in the growth of human breast cancer. Cancer Res. 51:3996–4000.[Abstract/Free Full Text]
  38. Mukherjee S. and Conrad S.E. (2005) c-Myc suppresses p21WAF1/CIP1 expression during estrogen signaling and antiestrogen resistance in human breast cancer cells. J. Biol. Chem. 280:17617–17625.[Abstract/Free Full Text]
  39. Singh R.P., Dhanalakshmi S., Tyagi A.K., Chan D.C., Agarwal C., Agarwal R. (2002) Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels. Cancer Res. 62:3063–3069.[Abstract/Free Full Text]
  40. Singh R.P., Mallikarjuna G.U., Sharma G., Dhanalakshmi S., Tyagi A.K., Chan D.C., Agarwal C., Agarwal R. (2004) Oral silibinin inhibits lung tumor growth in athymic nude mice and forms a novel chemocombination with doxorubicin targeting nuclear factor kappaB-mediated inducible chemoresistance. Clin. Cancer Res. 10:8641–8647.[Abstract/Free Full Text]
  41. Rodriguez-Burford C., Lubet R.A., Eto I., Juliana M.M., Kelloff G.J., Grubbs C.J., Steele V.E. (1999) Effect of reduced body weight gain on the evaluation of chemopreventive agents in the methylnitrosourea-induced mammary cancer model. Carcinogenesis 20:71–76.[Abstract/Free Full Text]
  42. Rodriguez-Burford C., Lubet R.A., Steele V.E., Eto I., Bandy M., Juliana M.M., Weiss H.L., Grizzle W.E., Kelloff G.J., Grubbs C.J. (2001) Effects of acute and chronic body weight gain reductions in the evaluation of agents for efficacy in mammary cancer prevention. Oncol. Rep. 8:373–379.[ISI][Medline]
  43. Gottardis M.M. and Jordan V.C. (1987) Antitumor actions of keoxifene and tamoxifen in the N-nitrosomethylurea-induced rat mammary carcinoma model. Cancer Res. 47:4020–4024.[Abstract/Free Full Text]
  44. Jordan V.C. (1987) Laboratory models of breast cancer to aid the elucidation of antiestrogen action. J. Lab. Clin. Med. 109:267–277.[ISI][Medline]
  45. Rose D.P., Fischer A.H., Jordan V.C. (1981) Activity of the antioestrogen trioxifene against N-Nitrosomethylurea-induced rat mammary carcinomas. Eur. J. Cancer Clin. Oncol. 17:893–898.[ISI][Medline]
  46. Medina D., Sivaraman L., Hilsenbeck S.G., Conneely O., Ginger M., Rosen J., Omalle B.W. (2001) Mechanisms of hormonal prevention of breast cancer. Ann. NY Acad. Sci. 952:23–35.[Abstract/Free Full Text]
  47. Russo I.H. and Russo J. (1998) Role of hormones in mammary cancer initiation and progression. J. Mammary Gland Biol. Neoplasia 3:49–61.[CrossRef][ISI][Medline]
  48. Hewitt S.C., Bocchinfuso W.P., Zhai J., Harrell C., Koonce L., Clark J., Myers P., Korach K.S. (2002) Lack of ductal development in the absence of functional estrogen receptor alpha delays mammary tumor formation induced by transgenic expression of ErbB2/neu. Cancer Res. 62:2798–2805.[Abstract/Free Full Text]
  49. Tyagi A.K., Agarwal C., Chan D.C., Agarwal R. (2004) Synergistic anti-cancer effects of silibinin with conventional cytotoxic agents doxorubicin, cisplatin and carboplatin against human breast carcinoma MCF-7 and MDA-MB468 cells. Oncol. Rep. 11:493–499.[ISI][Medline]
  50. Barzaghi N., Crema F., Gatti G., Pifferi G., Perucca E. (1990) Pharmacokinetic studies on IdB 1016, a silybin- phosphatidylcholine complex, in healthy human subjects. Eur. J. Drug Metab. Pharmacokinet 15:333–338.[ISI][Medline]
  51. Weyhenmeyer R., Mascher H., Birkmayer J. (1992) Study on dose-linearity of the pharmacokinetics of silibinin diastereomers using a new stereospecific assay. Int. J. Clin. Pharmacol. Ther. Toxicol. 30:134–138.[Medline]
  52. Davis-Searles P.R., Nakanishi Y., Kim N.C., Graf T.N., Oberlies N.H., Wani M.C., Wall M.E., Agarwal R., Kroll D.J. (2005) Milk thistle and prostate cancer: differential effects of pure flavonolignans from Silybum marianum on antiproliferative end points in human prostate carcinoma cells. Cancer Res. 65:4448–4457.[Abstract/Free Full Text]
Received January 20, 2006; revised March 10, 2006; accepted March 22, 2006.


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