Chemoprevention by curcumin during the promotion stage of tumorigenesis of mammary gland in rats irradiated with {gamma}-rays

Hiroshi Inano4, Makoto Onoda, Naoshi Inafuku1, Megumi Kubota1, Yasuhiro Kamada1, Toshihiko Osawa2, Hisae Kobayashi3 and Katsumi Wakabayashi3

First Research Group, National Institute of Radiological Sciences, 9-1 Anagawa-4-chome, Inage-ku, Chiba-shi 263-8555,
1 Ryukyu Bio-Resource Development Co. Ltd, 606-2 Toyohara, Motobu-cho 905-0204, Okinawa,
2 Laboratory of Food and Biodynamics, Nagoya University Graduate School of Bioagricultural Sciences, Furo-cho, Chikusa-ku, Nagoya-shi 464-8601 and
3 Institute for Molecular and Cellular Regulation, Gunma University, Showa-machi, Maebashi-shi 371-8512, Japan


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have evaluated the chemopreventive effects of curcumin on diethylstilbestrol (DES)-induced tumor promotion of rat mammary glands initiated with radiation. Sixty-four pregnant rats received whole body irradiation with 2.6 Gy {gamma}-rays from a 60Co source at day 20 of pregnancy and were divided into two groups after weaning. In the control group of 39 rats fed a basal diet and then implanted with a DES pellet for 1 year, 33 (84.6%) developed mammary tumors. Twenty-five rats were fed diet containing 1% curcumin immediately after weaning and received a DES pellet, as for the control. The administration of dietary curcumin significantly reduced the incidence (28.0%) of mammary tumors. Multiplicity and Iball's index of mammary tumors were also decreased by curcumin. Rats fed the curcumin diet showed a reduced incidence of the development of both mammary adenocarcinoma and ER(+)PgR(+) tumors in comparison with the control group. On long-term treatment with curcumin, body weight and ovarian weight were reduced, but liver weight was increased. Compared with the control rats, the curcumin-fed rats showed a significant reduction in serum prolactin, whereas estradiol-17ß and progesterone concentrations were not significantly different between the two groups. Curcumin did not have any effect on the concentration of free cholesterol, cholesterol ester and triglyceride. Feeding of the curcumin diet caused a significant increase in the concentrations of tetrahydrocurcumin, arachidonic acid and eicosapentaenoic acid and a significant decrease in thiobarbituric acid-reactive substance concentration in serum. Whole mounts of the mammary glands showed that curcumin yielded morphologically indistinguishable proliferation and differentiation from the glands of the control rats. These findings suggest that curcumin has a potent preventive activity during the DES-dependent promotion stage of radiation-induced mammary tumorigenesis.

Abbreviations: DES, diethylstilbestrol; ER, estrogen receptor; FSH, follicle stimulating hormone; LH, luteinizing hormone; LPS, lipopolysaccharide; MDA, malondialdehyde; NOS, nitric oxide synthase; PgR, progesterone receptor; TBARS, thiobarbituric acid-reactive substances.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epidemiological surveys suggest that diet has an impact on cancer incidence. Frequent consumption of vegetables and fruits decreases the risk for human cancer (13). Recently, attention has been focused on identifying dietary phytochemicals which have the ability to inhibit the processes of carcinogenesis. Extracts of plants or their fractionated ingredients were found to possess inhibitory effects against chemically induced carcinogenesis (4). Curcumin (Figure 1Go) is a major component of turmeric, the dried rhizome of Curcuma longa L. which is commonly used as a yellow coloring and flavoring agent in food in Asian countries. Commercial grade curcumin has shown anticarcinogenic activity in animals as indicated by ability to block colon tumor initiation induced by azoxymethane (5) and skin tumor promotion induced by phorbol ester (6). Furthermore, curcumin has been reported to possess anti-inflammatory activity and is a potent inhibitor of reactive oxygen-generating enzymes such as lipoxygenase/cyclooxygenase (7,8), xanthine dehydrogenase/oxidase (9) and nitric oxide synthase (NOS) (10,11). Lack of a mutagenic effect of curcumin was also reported in the presence or absence of a rat liver microsomal activation system in the Ames test with Salmonella typhimurium (12). Bhavanishankar et al. (13) found that an alcohol extract (including curcumin) of turmeric was non-toxic, although curcumin has been reported to inhibit the growth of a wide variety of tumor cells, whereas normal cells were found to be relatively resistant (14). In the present study, we have evaluated the chemopreventive effects of curcumin on diethylstilbestrol (DES)-induced tumor promotion of rat mammary glands initiated with radiation and the endocrinological and pharmacological activities of the agent are discussed.



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Fig. 1. Chemical structure of curcumin [chemical name 1,7-bis(4'-hydroxy-3'-methoxyphenyl)-1,6-heptadiene-3,5-dione].

 

    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials
Curcumin, commonly used in foods as a coloring agent, was obtained from Aldrich Chemical Co. (Milwaukee, WI). Diet containing 1% (w/w) curcumin was prepared in biscuit form by Funabashi Farm (Chiba, Japan). A basal diet (MB-1) of the same form was used for the control experiments. The major components of MB-1 are as follows: total carbohydrate, 54.1%; protein, 24.6%; fat, 4%; fiber, 3.8%; moisture, 7.7%; ash, 5.8%. DES, cholesterol and sulfatase were purchased from Sigma (St Louis, MO). ß-Glucuronidase was purchased from Wako Pure Chemical Industies Ltd (Osaka, Japan). Pellets were prepared in a medical grade Silastic tube (Dow Corning Co., Midland, MI) and were filled with 3 mg DES mixed with 27 mg cholesterol. [2,4,6,7-3H]Estradiol-17ß (sp. act. 4 TBq/mmol), [17{alpha}-methyl-3H]R5020 (sp. act. 3 TBq/mmol) and non-labeled R5020 (17{alpha},21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) were purchased from Du Pont/NEN Research Products (Boston, MA).

Animals and treatment
The rats used in the present study were treated and handled according to the Recommendations for Handling of Laboratory Animals for Biomedical Research compiled by the Committee on the Safety and Handling Regulations for Laboratory Animal Experiments in our Institute. Wistar-MS rats from a stock colony of Nippon SLC Co. (Hamamatsu, Japan) were kept at 23 ± 1°C in a controlled environment (14 h light/10 h dark). They received water and food ad libitum. Sixty-four pregnant rats received whole body irradiation with 2.6 Gy {gamma}-rays (0.15 Gy/min) from a 60Co source at day 20 of pregnancy (the presence of a vaginal plug denoting day 1) and were divided into two groups after weaning. Serving as the control group, 39 rats were fed a basal diet (MB-1) and were then implanted with a DES pellet at 1 month after weaning. Twenty-five rats were fed diet containing 1% curcumin immediately after weaning and received a DES pellet at 1 month after termination of nursing (Figure 2Go). The pellets were replaced every 8 weeks. The rate of release of DES from the pellet was 0.38 ± 0.01 µg/day (15). The rats were examined for palpable mammary tumors for 1 year starting from the date of pellet implantation. When mammary tumors >2 cm in diameter were detected, the rats were killed by CO2 asphyxiation and the tumors were removed. Each mammary tumor was divided into two portions: one portion was fixed in 10% neutral buffered formalin for histopathological examination and the other was trimmed off surrounding normal tissue and immediately frozen at -80°C until use for receptor assays. The remaining rats were killed 1 year after administration of the DES pellet and were autopsied to ascertain whether they had any non-palpable mammary tumors. Tumor incidence was calculated from the number of rats with tumors within 1 year. Iball's index of mammary tumors was calculated as follows: the ratio of incidence (%) to the average latency period in daysx100 (16).



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Fig. 2. Experimental schedule in this study. Open bar, control diet (MB-1); closed bar, diet containing 1% curcumin; open arrowhead, implantation with DES pellet; closed arrowhead, whole body irradiation with 2.6 Gy {gamma}-rays at day 20 of pregnancy; M, months old.

 
Assays
A blood sample was collected by cardiocentesis under anesthesia and serum prepared as usual. The sera were frozen immediately and stored at 80°C until the assay was started. For assays of total curcuminoids (free form plus conjugates), serum was incubated with 10 mM McIlvaine buffer (pH 5.0) containing 20% ascorbic acid, 0.17% EDTA, 500 U ß-glucuronidase and 40 U sulfatase at 37°C for 60 min (17). Curcumin and its metabolites were extracted with ethylacetate and then analyzed by HPLC with a multiwavelength detector on a Develosil ODS-HG-5 column (4.6x250 mm; Nomura Chemical Co., Seto, Japan) eluted with a mixture of acetonitrile:water (1:1 v/v) containing 0.1% trifluoroacetic acid as solvent system at a flow rate of 1 ml/min. The chromatogram was monitored at a wavelength of 430 nm for detection of curcumin and at 280 nm for tetrahydrocurcumin (18). Concentrations of prolactin, luteinizing hormone (LH) and follicle stimulating hormone (FSH) were determined with NIDDK radioimmunoassay kits (the National Hormone and Pituitary Program, Rockville, MD). Estradiol-17ß, progesterone, cholesterol and triglyceride were assayed by commercially available kits. Fatty acids were extracted from serum with hexane and were treated with 14% trifluorobenzene dissolved in methanol:methanol:benzene (35:35:30) for 10 min in boiling water for esterification. The methyl esters of fatty acids were analyzed by gas chromatography with a hydrogen flame ionization detector (19). For assay of lipid peroxidation products, the serum was mixed with 20% trichloroacetic acid and 0.67% thiobarbituric acid and then heated for 15 min in boiling water. The concentration of thiobarbituric acid-reactive substances (TBARS) extracted with n-butanol was estimated by absorption at 530 nm. TBARS were expressed as malondialdehyde (MDA) amounts, using freshly produced MDA as standard prepared from 1,1,3,3-tetramethoxypropane with HCl (20).

Whole mounts of mammary glands
After feeding of a diet containing curcumin for 1 year, the inguinal mammary glands were dissected from the inner surface of the skin, retaining as much of the connective tissue as possible, and spread and dried slightly on filter paper. After fixing in 10% formalin buffered with 0.1 M phosphate buffer (pH 7.2) and defatting with ethyl ether, the preparations were stained with alum carmine, destained in ethanol and stored in cedar oil (21).

Histological examination of mammary tumors
All mammary tumors were fixed immediately in 10% formalin buffered with 0.1 M phosphate buffer (pH 7.2). Each paraffin section (4 µm in thickness) was prepared and stained with hematoxylin and eosin. The tumors were classified as adenocarcinoma or fibroadenoma according to the criteria for the classification of rat mammary tumors (22).

Assay of steroid receptors
The tissues obtained from all mammary tumors >2.0 cm in diameter were homogenized in 10 mM Tris–HCl buffer (pH 7.4) containing 1.5 mM EDTA and 1 mM dithiothreitol. Homogenates were centrifuged at 105 000 g for 60 min at 4°C and the obtained cytosol fraction was used for assay of the estrogen receptor (ER) and progesterone receptor (PgR). The receptors were analyzed by a dextran-coated charcoal method using [2,4,6,7-3H]estradiol-17ß and [17{alpha}-methyl-3H]R5020, respectively, as radioligands (23,24). Maximum binding sites and dissociation constant (Kd) values for the receptors were determined by a Scatchard plot analysis (25).

Statistical analysis
Body weight, organ weight, tumor incidence, tumor multiplicity, latency period and biochemical parameters in serum were compared between the rats fed the control and curcumin diets. All statistical analyses were performed using StatView-J4.5 software (Abacus Concepts, Berkeley, CA). Tumor incidence, which is expressed as the percentage of rats with tumors, was analyzed statistically by {chi}2 test. The cumulative proportions of rats with tumors (incidence curves) were calculated by the product-limit method where rats which died or were killed without mammary tumors were included and the difference between groups was tested for statistical significance by the Mantel–Cox test. Numbers of adenocarcinomas and fibroadenomas in the groups were analyzed by Fisher's exact probability test. Tumor multiplicity, expressed as mean number of tumors/rat, was analyzed by unpaired t-test. Differences in body weight, organ weight, latency period and biochemical parameters between the groups were analyzed statistically by Student's t-test. Differences were considered statistically significant at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Prevention by curcumin of the development of mammary tumors
For control experiments, of the 39 pregnant rats which were administered whole body irradiation with 2.6 Gy {gamma}-rays at day 20 of pregnancy and then treated with DES after nursing, 33 (84.6%) rats developed mammary tumors during the experimental period of 1 year (Figure 3Go). The administration of dietary 1% curcumin together with DES implantation in the 25 experimental rats significantly decreased the incidence (28.0%) of total mammary tumors (P < 0.0001). The number of mammary tumors/tumor-bearing rat in the curcumin-fed group was half (NS, P = 0.092) that in the rats fed the control diet (1.9 ± 0.2). The Iball's index for overall development of mammary tumors in the curcumin-fed rats was also one-third of that in the control group (Table IGo). No significant difference in the latency period was observed between the control and curcumin-fed groups. The administration of dietary curcumin together with DES implantation in the irradiated rats significantly decreased the cumulative incidence curve (P < 0.0001) of mammary tumors for the 1 year period, compared with the control diet group (Figure 4Go). The appearance of the first palpable tumors was delayed by ~2.5 months in the curcumin-fed group compared with that in the control group.



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Fig. 3 . Incidence of mammary tumors in {gamma}-ray-irradiated rats. Bar, SD. The numbers in parentheses on the top of the bar represent the actual number of rats bearing tumors and rats used.

 

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Table I. Development of mammary tumors in rats fed control diet and curcumin diet
 


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Fig. 4 . Cumulative incidence of development of mammary tumors in irradiated rats treated with DES and curcumin. Statistical evaluation of the cumulative proportion data (incidence curves) by Mantel–Cox test yielded P < 0.0001, indicating a significant difference between the control diet and curcumin-containing diet groups. Solid and dotted lines represent control diet and curcumin diet groups, respectively.

 
Histological characterization of mammary tumors
All of the mammary tumors induced in the control group and curcumin-fed group were examined histologically. The proportion of adenocarcinomas and fibroadenomas in total tumors was 30.2 and 69.8%, respectively, in rats fed the control diet (Table IGo). By the administration of curcumin, the proportion (14.3%) of adenocarcinomas was decreased to half of that in the control group, conversely, one might say that the proportion (85.7%) of fibroadenomas was 1.2-fold higher than that in the control rats. However, no significant difference (P = 0.664) in the proportion of adenocarcinomas and fibroadenomas was observed between the two groups.

Biological effects of long-term treatment with curcumin
Body weight and organ weight values at the end of the experiment are summarized in Table IIGo. A significant reduction (P < 0.05) in body weight was observed in rats fed the diet containing 1% curcumin for 1 year in spite of similar calorie intake (P = 0.570) in both curcumin-fed (63.7 ± 1.1 kcal/rat/day) and control (62.6 ± 1.6 kcal/rat/day) groups. The weights of adrenals and uterus were decreased slightly by the administration of curcumin, but no significant difference was observed (P = 0.075 for adrenal and P = 0.086 for uterus). The treatment with curcumin increased liver weight significantly (P < 0.01), but ovarian weight was reduced markedly (P < 0.05). No change in weight of normal pituitary gland was observed between the control and curcumin-fed groups (P = 0.525). Interestingly, when the 39 control rats were autopsied, 22 (56.4%) were found to have developed pituitary tumors, while the pituitary tumor incidence (12.0%) in the curcumin-fed rats was one-fifth of that in the control rats (P < 0.0005).


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Table II. Biological effects of long-term administration of curcumin
 
Serum concentration of curcuminoids, hormones, lipids and TBARS
The serum concentrations of curcumin and its metabolites were measured 1 year after the administration of dietary curcumin together with DES implantation in the irradiated rats. Curcumin and tetrahydrocurcumin were estimated as 6.0 ± 2.0 and 112 ± 27 ng/ml serum, respectively (Table IIIGo). The serum estradiol-17ß concentration in the rats fed the curcumin diet was reduced to 52% of that obtained in rats fed the control diet, but no significant difference was observed between the two groups (P = 0.203). The progesterone concentration in the rats fed the curcumin diet was 1.3-fold higher than that in the control rats (P = 0.427). The serum prolactin concentration of the curcumin-fed rats was 80% less than that of control rats (P < 0.05). No changes in LH (P = 0.631) and FSH (P = 0.410) concentrations were observed in the curcumin-fed rats. Also, curcumin did not have any effect on the concentrations of free cholesterol (P = 0.249), cholesterol ester (P = 0.188) and triglyceride (P = 0.409). The serum concentration of TBARS was significantly decreased by long-term treatment with curcumin (P < 0.05).


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Table III. Effect of curcumin on serum concentrations of hormones, lipids, TBARS and curcumin
 
Fatty acid profile in serum
The serum concentrations of fatty acids were measured 1 year after the start of the administration of dietary curcumin together with DES implantation in the irradiated rats (Table IVGo). With regard to unsaturated fatty acids, the serum concentrations of arachidonic acid (P < 0.05) and eicosapentaenoic acid (P < 0.01) in rats fed the curcumin diet were significantly higher than in rats in the control group. However, no significant change was observed in concentrations of the other fatty acids such as oleic acid (P = 0.797), linoleic acid (P = 0.114), linolenic acid (P = 0.894) and docosahexanoic acid (P = 0.658). The sum of the polyunsaturated fatty acids (>= three unsaturated bonds) was increased significantly (P < 0.05) by treatment with curcumin and total unsaturated fatty acids (>= one unsaturated bond) was not affected by curcumin (P = 0.427). With regard to saturated fatty acids, the serum stearic acid concentration in rats fed the curcumin diet was 1.3-fold higher than that in control rats, but no significant difference was observed between the two groups (P = 0.071). Fatty acid ratios of total saturated fatty acids to total unsaturated fatty acids (S/U) and of total saturated fatty acids to total polyunsaturated fatty acids (S/PU) were not significantly altered by long-term administration of curcumin.


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Table IV. Effect of curcumin on the fatty acid profile in serum
 
Effect of curcumin on development of mammary glands
At the end of the experiment, whole mounts of inguinal mammary glands were prepared to examine the effects of curcumin on the development and differentiation of the glands by long-term treatment. In the irradiated rats fed the control diet, mammary glands showed many alveolar buds with branched lactiferous ducts due to the DES implantation (Figure 5a and bGo). The whole mounts (Figure 5c and dGo) showed that the mammary glands in rats fed curcumin diet exhibited less proliferation and differentiation than those of the glands of control rats, because both number and size of alveolar buds were small.



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Fig. 5. Whole mount observation of inguinal mammary glands of rats. (a and b) Control rats fed basal diet (MB-1); (c and d) rats fed diet containing 1% curcumin. Scale bars: a and c, 3 mm; b and d, 1 mm.

 
ER and PgR in mammary tumors
Table VGo shows the results of analysis of ER and PgR in the cytosol fraction obtained from homogenates of mammary tumors >2.0 cm in diameter. Many (74.2%) of the mammary tumors that developed in rats fed the control diet were ER(+)PgR(+) and only one tumor (3.2%) was ER(–)PgR(–). Conversely, rats fed the curcumin diet showed increased numbers (57.1%) of ER(–)PgR(–) tumors and decreased numbers (14.3%) of ER(+)PgR(+).


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Table V. ER and PgR in mammary tumors
 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In our previous study, it was found that whole body irradiation of pregnant rats results in a higher incidence of mammary tumors than that observed in irradiated virgin rats (26) and we suggested that the mammary cells in the differentiated glands of pregnant rats are particularly susceptible to radiation. Although numerous proliferated epithelial cells in the glands are removed rapidly by the involution process after weaning, the origin of the mammary tumors initiated by radiation during pregnancy appears to be the cells that are not removed during involution, which proliferate in the presence of DES as the tumor promoter. Reddy and Aggarwal (14) have reported that curcumin could inhibit the growth of tumor cells, whereas normal cells are found to be relatively resistant. We would suggest that one possible mechanism in the chemoprevention of mammary tumors by long-term treatment with curcumin is inhibition of the proliferation of preneoplastic cells or tumor origin cells initiated by radiation.

Data presented herein indicate that curcumin markedly reduces tumorigenesis in mammary glands in irradiated rats during DES-dependent promotion. Curcumin has been shown to display antitumor properties in animals, as indicated by its ability to prevent tumorigenesis in skin (27), colon (28) and oral cavity (29) induced by chemical carcinogens. In the 7,12-dimethylbenz[a]anthracene-induced mammary tumor model in rats, when curcumin was administered by i.p. injection at 100 mg/kg body wt (30) or in a diet containing 1% curcumin (5) prior to dosing with the chemical carcinogen, the incidence of animals with tumors was not significantly altered. However, administration of a concentration twice as high as the above doses of curcumin slightly decreased tumor incidence to 85% of the control group. Therefore, our results are the first to show that curcumin markedly inhibits tumorigenesis in mammary glands. Also, incidence (12.0%) of pituitary tumors in curcumin-fed rats indicated less than half of that (28.0%) in the control group. However, it is inconclusive whether the reduced incidence was caused by an antipromotion effect of curcumin, because of a lack of data on experimental groups given DES without radiation and with or without curcumin in the present study. Further research is needed to establish the mechanism of prevention of pituitary tumors by curcumin.

Yokoro et al. (31) have reported that rat mammary tumorigenesis was mediated through estrogenic stimulation of prolactin release from the pituitary gland. In our current study, chemoprevention by curcumin of mammary tumor promotion by DES was associated with a significant decrease (20% of the control value) in serum concentration of prolactin in rats fed the curcumin diet. This result is consistent with other reports in which inhibition of mammary gland DNA synthesis by 2-bromo-{alpha}-ergocryptine-induced prolactin suppression resulted in a decrease in mammary tumorigenesis in rats (32). Also, in previous works, a decrease in prolactin concentration and chemoprevention of radiation-induced mammary tumors resulted from administration of either dietary dehydroepiandrosterone (33) or bezafibrate (34) in the presence of DES implantation.

Holder et al. (35) and Ravindranath and Chandrasekhara (36) have reported the absorption, metabolism and excretion of curcumin administered orally. Curcumin was metabolized to tetrahydrocurcumin during absorption through the intestine. Almost all the curcuminoids were conjugated to the glucuronide and/or sulfate forms in liver and excreted mainly in the feces after enterohepatic circulation of the metabolites. Using rats fed a diet containing 1% curcumin for 1 year in the present experiment, serum concentrations (free form plus conjugates) of curcumin and its metabolites were measured. Concentration of tetrahydrocurcumin was ~20-fold higher than that of curcumin. Tetrahydrocurcumin exhibited a significant inhibitory effect on 12-O-tetradecanoylphorbol-13-acetate-induced superoxide anion radical generation (37) and a greater inhibitory effect on lipid peroxidation of erythrocyte membrane ghosts induced by t-butylhydroperoxide than curcumin (18,38). It was shown that feeding a diet containing 0.5% tetrahydrocurcumin resulted in a significant repression of 1,2-dimethylhydrazine-induced formation of aberrant crypt foci, which are regarded as a precursor lesion for colon cancer (39). Tetrahydrocurcumin may have the same important physiological and pharmacological properties as curcumin in vivo due to the ß-diketone moiety as well as phenolic hydroxy groups.

An increase in peroxidase activity in the mammary glands is observed to accompany sexual maturity (40). Recently, it was reported by Roy et al. (41) that oxidation of DES by the peroxidase activity of cytochrome P-4501A1 produces some reactive intermediates, presumably the semiquinone and quinone of DES, and curcumin is a potent inhibitor of cytochrome P-4501A1 (42). Superoxide anion radicals are generated by redox cycling between DES and its quinone (43) and reduce iron, which in turn reduces hydrogen peroxide, the secondary product of one-electron auto-oxidation of superoxide anion radicals, to hydroxyl radicals (44,45). The unsaturated fatty acids present in the cell membrane are susceptible to hydroxyl radicals, which are much more reactive than superoxide anion radicals. Peroxidation of polyunsaturated fatty acids containing three or more double bonds by oxygen free radicals induces them to produce lipid hydroperoxides or TBARS, such as MDA. In this paper, the concentrations of arachidonic acid and eicosapentaenoic acid during ingestion of the curcumin diet were higher than those in rats fed the control diet and the amounts of TBARS derived from hydroperoxidized fatty acids induced by DES were also significantly reduced by curcumin. These findings suggest that curcumin inhibits peroxidation of polyunsaturated fatty acids in the presence of DES. Huang et al. (7,8) also showed that the antitumor promotion activity of curcumin is associated with suppression of arachidonic acid metabolism. Arachidonic acid metabolism is known to produce free radicals in living cells. Curcumin appears to be a potent scavenger of oxygen free radicals, such as superoxide anion radicals (37,46) and hydroxyl radicals (47,48). Its antioxidant activity is further shown by its capacity to inhibit lipid peroxidation. For instance, curcumin suppresses the lipid peroxide levels in serum and liver of rats irradiated with {gamma}-rays (49) and of renal epithelial (LLC-PK1) cells incubated with hydrogen peroxide (50). Lipid peroxides are toxic to cells because they decrease membrane fluidity and permeability and induce DNA damage and, ultimately, result in cell injury (51). Shalini and Srinivas (52) have shown that curcumin inhibits lipid peroxide-induced DNA damage. The role of free radicals in the tumor promotion stage has been reviewed (53,54). From the above findings, curcumin may scavenge the free radicals derived from DES metabolism and exhibit antipromotion activity. Our observations support the hypothesis that one aspect of the antitumor activity of curcumin during the promotion stage may be linked to reduction of free radicals.

Furthermore, our results demonstrate that the serum concentration of eicosapentaenoic acid in the rats fed the curcumin diet was ~2-fold higher than that in control rats. Eicosapentaenoic acid is an {omega}-3 polyunsaturated fatty acid. Growth of a human breast cancer cell line (MDA-MB-231) (55) and of a rat mammary adenocarcinoma (R3230AC) (56) is suppressed by {omega}-3 polyunsaturated fatty acids. Recently, it was reported that dietary {omega}-3 polyunsaturated fatty acids reduce the activities and levels of both HMG-CoA reductase (57) and cyclooxygenase-2 (58) in rat mammary glands. Inhibition of HMG-CoA reductase is known to suppress a post-translational processing of p21ras (59) and prevent tumorigenesis in mammary glands (60) and colon (61). Inhibition of p21ras processing may inhibit malignant transformation (62). Cyclooxygenase-2 is usually not expressed in various organs, but its expression in certain cell types can be rapidly induced by mitogens and hormones (63). Intake of the cyclooxygenase-2 inhibitor nabumetone during the time corresponding to the post-initiation phase has a chemopreventive effect on N-methyl-N-nitrosourea-induced mammary carcinogenesis in rats (64). In addition, reduced expression of cyclooxygenase-2 in the mammary glands of rats fed {omega}-3 polyunsaturated fatty acids was accompanied by a decreased level of p21ras protein (58). Therefore, it is likely that prevention of mammary carcinogenesis may occur in curcumin-fed rats having increased concentrations of serum {omega}-3 polyunsaturated fatty acids, such as eicosapentaenoic acid.

Also, dietary {omega}-3 polyunsaturated fatty acids reduced the activity of NOS in lipopolysaccharide (LPS)-stimulated macrophage cells (65). In rat mammary glands, certain isoforms of NOS localize to myoepithelial cells of the glandular epithelium and marked expression of i-NOS is observed after exposure to LPS, an inflammatory agent (66). Excessive NO produced in inflamed tissues could contribute to the process of carcinogenesis (67,68). Chan et al. have found that curcumin reduced NO production in LPS- and interferon-{gamma}-stimulated peritoneal cells (69). This may result from one or more mechanisms: reduction of i-NOS gene expression (11) and/or scavenging of NO molecules (70). Thus, inhibition of NO synthesis by NOS inhibitors gives reduced growth of rat mammary adenocarcinoma (71).

Finally, Gross and Dreyfuss (72,73) have reported that the incidence of radiation-induced tumors in rats was significantly reduced by calorie restriction. In our present experiments, rats fed the curcumin diet had a significant reduction in body weight. However, food intake was not reduced by chronic curcumin treatment. Body weight in rats fed the curcumin diet was reduced to 93% of that observed in rats fed the control diet. The reduction is statistically significant, but would be relatively low for prevention of the development of radiation-induced mammary tumors. From the above-mentioned, it is likely that a reduction in body weight in rats fed the curcumin diet did not contribute to the prevention of development of mammary tumors.

In conclusion, the DES-dependent promotion of radiation-induced mammary tumorigenesis was markedly inhibited by administration of dietary curcumin. The mechanism of chemoprevention involves both endocrinological and pharmacological effects of curcumin during tumor development in the radiation-initiated cells. These results provide useful information for further development of compounds as chemopreventive agents for human clinical trials.


    Acknowledgments
 
This work was partly supported by a project research grant for Experimental Studies on Radiation Health, Detriment and its Modifying Factors and also by a grant from the Special Program for Bioregulation of the National Institute of Radiological Sciences. We thank Dr J.Ueda for valuable discussions concerning antioxidant activity of curcumin and Mrs M.Takahashi for excellent assistance in the care of the animals.


    Notes
 
4 To whom correspondence should be addressed Email: inano{at}nirs.go.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received October 20, 1998; revised December 29, 1998; accepted February 11, 1999.