Prevention of radiation-induced mammary tumors

Int. J. Radiation Oncology Biol. Phys., Vol. 52, No. 1, pp. 212–223, 2002 Copyright © 2002 Elsevier Science Inc. Printed in the USA. All rights reserved 0360-3016/02/$–see front matter

PII S0360-3016(01)02651-7

BIOLOGY CONTRIBUTION

PREVENTION OF RADIATION-INDUCED MAMMARY TUMORS HIROSHI INANO, PH.D.,

AND

MAKOTO ONODA, PH.D.

Redox Regulation Research Group, Research Center for Radiation Safety, National Institute of Radiological Sciences, Inage-ku, Chiba-shi, Japan The radiation-induced rat mammary tumor model is useful for studying tumor prevention by treatment in the initiation or promotion stage. In anti-initiation experiments, the administration of radical scavengers or spintrapping agents before or immediately after irradiation reduced the incidence of mammary tumors, suggesting that free radicals produced by exposure are a potent initiator. To evaluate the role of nitric oxide (NO) in the initiation, NO-specific scavengers or NO synthase inhibitors were administered during the initiation. These agents partially prevented the tumorigenesis, suggesting that radiation-induced NO contributes to tumor initiation. The administration of curcumin during irradiation reduced the incidence of the tumors in the presence of tumor promotor. In anti-promotion experiments on preventing diethylstilbestrol (DES)-dependent tumor development from mammary primodial cells exposed to radiation, tamoxifen decreased the tumor incidence. From the results, estrogen itself or prolactin induced by estrogen may be a promoter for the tumorigenesis. Bezafibrate and simvastatin, agents inducing hypolipidemia and hypocholesterolemia respectively, cause a decrease in the DES-dependent promotion of radiation-induced tumorigenesis. The simultaneous administration of curcumin and DES significantly reduces the development of mammary tumors in irradiated rats. In this review, the endocrinologic and pharmacologic significance of the anti-initiation and anti-promotion is discussed. © 2002 Elsevier Science Inc. Mammary tumor, Prevention, Anti-initiation, Anti-promotion.

of chemical carcinogen–induced mammary tumorigenesis by synthetic agents or natural products (6 –9), but none for radiation-induced tumors. Because the prevention of radiation-induced tumors is a matter of interest, this review focuses on recent concepts in the prevention of mammary tumorigenesis initiated with radiation and then promoted with synthetic estrogens. In addition, we propose appropriate strategies for future clinical chemoprevention trials of radiation-induced tumors.

INTRODUCTION Radiation is being used increasingly for medical and occupational reasons and is a cause of mammary tumors, leukemia, and other tumors in both humans and experimental animal models. Previous studies in our laboratory have demonstrated that irradiation of developed mammary glands in rats during pregnancy or lactation induces mammary tumors at a higher incidence than in irradiated virgin rats in the presence of a tumor promoter (1–3). The susceptibility of the mammary glands to radiation depends on estrogenstimulated development in pregnancy (2) and on prolactinstimulated differentiation in lactation (3). It is suggested that estrogen is a direct or indirect sensitizer for tumor initiation by radiation (4) and for promotion of tumorigenesis from primordial cells initiated with radiation (5). Recently, external sources of estrogen have become widely used. Oral contraceptive and hormone replacement therapy are two of the main sources of exogenous estrogen in women. There have been several reviews of the prevention

ANTI-INITIATION Ovariectomy before irradiation In the course of sexual maturation, the mammary glands of female animals develop to ensure lactation after childbirth. This development occurs in several phases; each phase is characterized by different anatomic and histologic features, and is influenced by different hormones. Ovarian hormones and pituitary hormones are the major hormones as a grant for Special Program on Bio-regulatory Mechanism from the Japan Science and Technology Agency. Acknowledgments—We are indebted to Drs. K. Wakabayashi, K. Suzuki, H. Kobayashi, H. Ishii-Ohba, H. Yamanouchi, N. Inafuku, M. Kubota, Y. Kamada, T. Osawa, and J. Ueda for their valuable advice and cooperation, and to K. Ikeda and M. Takahashi for their technical assistance. Received Jun 29, 2001. Accepted for publication Sep 4, 2001.

Reprint requests to: Dr. Hiroshi Inano, Redox Regulation Research Group, Research Center for Radiation Safety, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inageku, Chiba-shi 263-8555, Japan. Tel: ⫹81-43-206-3127; Fax: ⫹8143-255-6819; E-mail: [email protected] Supported by a research grant, “Radiation-induced Mammary Tumorigenesis,” from the National Institute of Radiological Sciences, and a projected research grant, “Experimental Studies on Radiation Health, Detriments and its Modifying Factors,” as well 212

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Table 1. Prevention of radiation-induced mammary tumorigenesis by ovariectomy before or after maturation* Rats with tumors Ovariectomy Nonovariectomy Ovariectomy at 70 days of age Ovariectomy at 23 days of age

No. of tumors

No. of rats used

No.

%

AC

FA

No. of tumors per tumor-bearing rat

20

6

30

2

4

1.0 ⫾ 0.0

30

9

30

7

10

1.9 ⫾ 0.4

23

6

26

1

5

1.0 ⫾ 0.0

Abbreviations: AC ⫽ adenocarcinoma; FA ⫽ fibroadenoma. * All rats were treated with DES, a tumor promoter, for 1 year after irradiation.

involved in development of the mammary glands. Estrogen promotes terminal end-bud development and duct elongation in mammary glands; progesterone promotes duct enlargement and ductule formation and growth (10). The synergistic effect of ovarian hormones and prolactin in the development of mammary glands has also been implicated in tumorigenesis in the mammary glands (11). When female rats of the Wistar-MS strain, were ovariectomized bilaterally after sexual maturation and then irradiated with 2.6 Gy ␥-rays to evaluate the role of ovarian hormones in the sensitivity to the initiation of mammary tumorigenesis by radiation (12), the tumor incidence of the ovariectomized rats was 30% in the presence of a tumor promoter, diethylstilbestrol (DES) (Table 1). This incidence is comparable to that observed in nonovariectomized virgin rats treated with both irradiation at the same dose and DES (2, 3, 13). In this experiment, the mammary glands of the ovariectomized rats had been exposed to estrogens synthesized in ovaries before the ovariectomy. Therefore, radiation-induced initiation of mammary tumorigenesis is not suppressed completely by ovariectomy after maturation. On the other hand, when immature female rats were ovariectomized bilaterally before the onset of the estrous cycle, the mammary glands demonstrated a low degree of differentiation with narrow ducts in adulthood. By bilateral ovariectomy before puberty and then irradiation with ␥-rays in adulthood, a relatively low incidence (26%) of mammary tumors was observed during the 1-year period of DES implantation (4). The incidence does not differ much from that in irradiated rats ovariectomized bilaterally after maturation. The mammary glands of adult rats ovariectomized before the onset of the estrous cycle may be exposed to estrogens synthesized by aromatase in extraovarian tissues, such as adiposites (14, 15), during the period up until tumor initiation with irradiation. Calorie restriction Recent studies have demonstrated that the prevention of chemical-induced mammary carcinogenesis by dietary restriction is due specifically to a reduction in calorie intake and not to a reduction in the intake of nutrients (16, 17). After consecutive total-body irradiation of 1.5 Gy ␥-rays five times at weekly intervals in Sprague-Dawley female rats, the incidence of tumors, most frequently of the mam-

mary glands, was 89%. A dietary restriction of 36% decreased to 23% the incidence of radiation-induced tumors (18). The rats fed a restricted diet lost body weight, but were in good health, physically active, and lived longer than the control rats maintained on a full diet (18). Also, calorie restriction caused an inhibition of development of X-rayinduced or ␥-ray-induced leukemia in mice (19, 20) and N-methyl-N-nitrosourea (MNU)-induced mammary tumors in rats (21, 22). Yoshida et al. have suggested that calorie restriction affects the initiation phase as well as the promotion phase of radiation-induced leukemogenesis (20). Evidence is accumulating of a specific effect of calorie restriction on the expression of oncogenes (23) and tumor suppressor genes (24) that are involved in the carcinogenic process. Meanwhile, 40% calorie restriction significantly reduced metabolites of oxidative DNA damage, such as 5-hydroxymethyluracil and 8-hydroxy-2⬘-deoxyguanosine (8-OHdG), in mammary gland and liver (25, 26). Calorie restriction in human subjects also has been shown to decrease urinary oxidized DNA (27). Rao et al. have shown that calorie restriction increases the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (28). Oxidative DNA damage levels will reflect the oxidant level, free radical detoxification ability, and DNA repair. Spin-trapping agent Spin-trapping agents such as nitrone derivatives have been shown to have pharmacologic activity in many ageassociated diseases (29). ␣-Phenyl-tert-butyl nitrone (PBN) is shown to react with free radicals to form a spin-adduct that is a relatively stable nitroxide free radical. Irradiation with ␥-rays in the presence of PBN resulted in a substantial reduction in the 8-OHdG produced by hydroxyl free radical attack on the DNA base 2⬘-deoxyguanosine (30). Also, PBN was effective in reducing liver nitric oxide (NO) generation elevated by lipopolysaccharide (LPS) administration (31). Miyajima and Kotake have reported that PBN inhibits the expression of inducible nitric oxide synthase (iNOS) mRNA (31) and protein (32). As shown in Table 2, the administration of PBN 30 min before irradiation with ␥-rays (1.5 Gy) during lactation, a period of high susceptibility to radiation, suppressed partially the incidence of mammary tumors in rats (33). From these results, it is suggested that radiation-

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Table 2. Anti-initiation activity of PBN, aminothiols, NO-related compounds, and curcumin in radiation-induced mammary tumorigenesis*

Treatment during initiation stage Control PBN (40 mg/rat, i.p.) Control WR-2721 (50 mg/rat, i.p.) Cysteamine (25 mg/rat, i.p.) Control WR-2721 (50 mg/rat, i.p.) Cysteamine (25 mg/rat, i.p.) Control WR-2721 (50 mg/rat, i.p.) Cysteamine (25 mg/rat, i.p.) Control WR-2721 (50 mg/rat, i.p.) Cysteamine (25 mg/rat, i.p.) Control 1,4-PB-ITU (50 ppm in drinking water) Fe(DETC)2 (DETC, 125 mg/rat, i.p. and FeSO4, 9.4 mg ⫹Nacitrate, 47 mg/rat, s.c.) Control Curcumin (1%, in diet)

Treatment for initiation 1.5 Gy at Day 21 of lactation 1.5 Gy at Day 20 of pregnancy 2.6 Gy at Day 20 of pregnancy 1.5 Gy at Day 21 of lactation 2.6 Gy at Day 21 of lactation 1.5 Gy at Day 21 of lactation

1.5 Gy at Day 20 of pregnancy

Rats with tumors

No. of tumors

No. of rats used

No.

%

AC

FA

No. of tumors per tumor-bearing rat

20 21 21 21 24 26 23 21 20 23 25 28 23 22 20

17 5 15 5 5 24 10 14 17 12 10 27 22 20 17

85 24 71 24 21 92 44 67 85 52 40 96 96 91 85

12 0 7 2 2 17 3 6 11 7 1 16 18 22 12

10 5 18 7 4 21 15 15 15 8 11 16 28 32 10

1.3 ⫾ 0.2 1.0 ⫾ 0.0 1.6 ⫾ 0.2 1.8 ⫾ 0.8 1.2 ⫾ 0.2 1.6 ⫾ 0.4 1.8 ⫾ 0.2 1.6 ⫾ 0.9 1.5 ⫾ 0.3 1.2 ⫾ 0.1 1.2 ⫾ 0.1 1.2 ⫾ 0.1 2.1 ⫾ 0.3 2.8 ⫾ 0.4 1.3 ⫾ 0.2

20

7

35

2

5

1.0 ⫾ 0.0

23 27 27

5 19 5

22 70 18

1 9 1

4 19 5

1.0 ⫾ 0.0 1.5 ⫾ 0.2 1.2 ⫾ 0.2

* All rats were treated with DES, as a tumor promoter, for 1 year after irradiation.

induced tumorigenesis depends on the level of oxygen free radicals or NO in a cell. Aminothiol derivatives The generation of oxygen radical species after exposure to radiation is considered one of the most important mechanisms of radiation-induced carcinogenesis. Cysteamine and S-2-(3-aminopropylamino)-ethylphosphorothioic acid (WR-2721) are aminothiol derivatives and potent radioprotectors. WR-2721 is a prodrug that requires dephosphorylation catalyzed by alkaline phosphatase in the plasma membrane (34) to generate the free thiol form, 2-(aminopropylamino) ethanethiol (WR-1065), as the active form of a radioprotector. The protection against the effects of radiation offered by WR-2721 and cysteamine is considered to be due to the scavenging of free radicals produced by the interaction of radiation and biologic molecules (35). However, little information is available on the in vivo effects of these agents on the development of radiation-induced tumors. There are only four reports that radiation-induced sarcoma (36), lymphoreticular tumors (37) and mammary tumors (38, 39) were suppressed by administration of WR2721 before ␥-ray irradiation. The administration of WR2721 30 min before irradiation with 1.5 Gy in Wistar-MS rats during pregnancy, a period of high susceptibility to radiation similar to lactation, suppressed the incidence of mammary tumors, which is 71% in saline-injected control rats, to 24% in the presence of a tumor promoter, DES (38). As shown in Table 2, treatment of pregnant rats with

cysteamine before whole body irradiation with 1.5 Gy also decreased the incidence (21%) of mammary tumors over a 1-year period. Tumor prevention by either agent is less effective at the higher dose of 2.6 Gy. Also, the high incidence (85%) of mammary tumors in rats irradiated at Day 21 of lactation (which is similar to the susceptibility to radiation of mammary glands of pregnant rats) and then administered with DES as promoter, was decreased to 52% by treatment with WR-2721 before the irradiation (39). There was a significant prolongation of the average latent period until the appearance of mammary tumors, when rats were injected with WR-2721 or cysteamine before irradiation with 1.5 Gy during pregnancy, but not with 2.6 Gy. No significant difference was observed among the rats irradiated with 1.5 or 2.6 Gy in the number of mammary tumors per tumor-bearing rat. Many of the mammary tumors that developed in the control rats were of the ER⫹PgR⫹ type. Administration of WR-2721 produced no tumors of the ER⫹PgR⫹ type. Cysteamine treatment potentiated the development of ER-negative tumors. WR-2721 and cysteamine had no effect on the serum concentration of prolactin in irradiated rats. The serum concentration of progesterone was significantly higher in irradiated rats treated with WR-2721 than in the control rats, but the estradiol concentration was reduced by treatment with WR-2721. The modes of action of WR-2721 proposed to date include induction of intracellular hypoxia by oxidation of the thiol moiety of the WR-1065 molecule (40). Moreover,

Prevention of radiation-induced tumor

protection of mammalian cells from the cytotoxic effects of ␥-rays by WR-1065 is accompanied by a substantial reduction in the yield of single- and double-strand breaks in the DNA (41– 43). Administration of cysteamine rapidly depleted serum prolactin by inhibition of dopamine-␤-hydroxylase (44). Because the action of prolactin in the mammary glands is related to a higher incidence of mammary tumors induced by radiation (3), the reduction in the prolactin level at the time of irradiation is one possible mechanism of the preventive action of cysteamine. On the other hand, when female Sprague-Dawley rats received S-2-aminoethylisothiouronium 䡠 Br 䡠 HBr (AET), another radioprotector, before partial body exposure to 4 Gy X-rays, the incidence and total number of mammary tumors did not decrease (45). The doses of radiation and radioprotector and the interval between the administration of radioprotector and the exposure are considered to be important factors that influence radioprotection. NO scavengers and NOS inhibitors Nitric oxide, a potentially toxic gas with free radical properties, acts as a messenger mediating various physiologic functions (46). In rat mammary glands, certain isoforms of NO synthase (NOS) localize to myoepithelial cells of the glandular epithelium, and marked expression of iNOS has been observed after exposure to an inflammatory agent, LPS (47). The enhancement of NO production by irradiation is attributed to high levels of expression of iNOS (48). Excessive production of NO by activated iNOS may result in the formation of toxic intermediates, such as peroxynitrite (ONOO⫺), causing tissue damage and genotoxicity (49), and thus have potential carcinogenic effects (50). In human breast cancer, iNOS is detected in peritumoral spindle cells in the stroma (51). Also, the level of iNOS protein or iNOS activity has been positively correlated with the degree of malignancy in a number of human breast cancers (51). The effect of 1,4-PB-ITU, S,S⬘-1,4-phenylene-bis(1,2ethanediyl) bis-isothiourea, on the suppression of iNOS and cyclooxygenase-2 (COX-2) is highly selective and pronounced (52). On administration of 1,4-PB-ITU to lactating rats for 6 days from 3 days before irradiation with 1.5 Gy ␥-rays to 3 days, the incidence (30%) of mammary tumors was significantly reduced in comparison with that (85%) of the control group (Table 2) (53). Also, the iron(II)-diethyldithiocarbamate complex, Fe (DETC)2, has been used to specifically scavenge NO formed in animal tissues in vivo from exogenous and endogenous sources (54). Lactating rats received whole body irradiation with 1.5 Gy ␥-rays from a 60Co source at weaning, and were injected simultaneously with DETC, i.p. and Fe2⫹-citrate, s.c. at 0, 8, and 24 h after irradiation. The high incidence (85%) of mammary tumors in control rats irradiated at weaning was decreased to 22% by treatment with Fe (DETC)2 during initiation with ␥-rays (53). Therefore, the radiation-induced initiation of mammary tumorigenesis may be partly caused by excessive NO produced by radiation-induced iNOS or a

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toxic intermediate, such as ONOO⫺, produced from a rapid reaction of NO with O⫺ 2 . Also, mammary tumor– derived NO promotes growth and metastasis by multiple mechanisms such as stimulation of tumor cell migration, invasiveness, and angiogenesis (55). In fact, inhibition of the synthesis of NO by NOS inhibitors leads to reduced growth of rat mammary adenocarcinoma (56). Curcumin Epidemiologic studies suggest that diet has an impact on the incidence of cancer. Frequent consumption of vegetables and fruits decreases the risk for human cancer. Recently, attention has been on idenfying dietary phytochemicals, which have the ability to inhibit the processes of carcinogenesis. Extracts of plants or their fractionated ingredients have been found to possess inhibitory effects against chemically induced carcinogenesis (57). Curcumin, 1,7-bis(4⬘-hydroxy-3⬘-methoxyphenyl)-1, 6heptadiene-3,5-dione, is a major component of turmeric, the dried rhizome of Curcuma longa L. which is commonly used as a yellow coloring in food, and has been reported to possess anti-inflammatory activity. As shown in Fig. 1, curcumin is a potent inhibitor of radical-generating enzymes, such as cyclooxygenase (COX) (58, 59), NOS (60, 61), and xanthine oxidase (XOD) (62), and also enhances the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPX) (63). Explanations of the cytotoxic effects of radiation have previously emphasized the involvement of reactive oxygen species such as the superoxide anion and hydroxyl radicals (64, 65). McLennan et al. have reported that superoxide anion radical is nontoxic, but a precursor in the formation of hydroxyl radical, which is the most toxic radical resulting from radiation (66). Curcumin is a unique compound, having both phenolic and ␤-diketone functional groups, and would be expected to have remarkable antioxidant and free radical scavenging activities (67, 68). During the feeding of a diet containing 1% curcumin between Day 11 of pregnancy and parturition, Day 23 of pregnancy, rats received whole body irradiation with 1.5 Gy ␥-rays from a 60Co source at Day 20 of pregnancy, and were then implanted with a DES as a tumor promoter 1 month after weaning. As shown in Table 2, the administration of dietary curcumin in the initiation stage reduced significantly the incidence (18%) of mammary tumors in comparison with that (70%) of rats fed the control diet (69). By histologic examination, the proportion of adenocarcinomas (17%) in total tumors in the curcumin-fed rats was found to be half of that (32%) in the control group. Curcumin does not have any toxic effect on organ weight or on the development and differentiation of mammary glands of pregnant rats. In addition, the serum concentrations of estradiol-17␤ and prolactin in curcumin-fed rats were retained at the control level. That there was no effect on litter size and body weight of pups born from curcumin-fed rats indicates the absence of toxicity of curcumin (69). Also, a lack of mutagenic effect

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Fig. 1. Hypothetical mechanisms of prevention of radiation-induced tumor initiation by curcumin. Curcumin blocks the radiation-induced activation of nuclear factor kappa B (NF-kB) by inhibiting inhibitory protein ␬B (I-kB) kinase activity and then suppresses the transcriptional activation of NF-kB-dependent genes of free radical generating enzymes (COX-2, iNOS, and xanthineoxidase [XOD]) and inflammatory cytokines (interleukin[IL]-6 and IL-8) (2). Also, curcumin enhances the expression of antioxidant enzymes (SOD, catalase, heme oxygenase [HO-1], and glutathione peroxidase[GPX]) (1) and scavenges directly free radicals produced by interaction of radiation and biomolecules. The anti-initiation action of curcumin could be mediated predominantly by anti-inflammation causing a decrease of free radicals.

of curcumin was also reported in the presence and absence of a rat liver microsomal activation system in the Ames test with Salmonella typhimurium (70). These results suggest that curcumin does not have any side effects. We suggest that one possible mechanism of the anti-initiation activity of curcumin is the scavenging of free radicals produced by radiation as a tumor initiator at target sites. NO plays a key role in physiologic as well as pathologic processes, including inflammation and cancer. In immunohistochemical experiments, the expression of iNOS in mammary glands treated with LPS was apparently decreased by curcumin treatment (48, 71). Furthermore, NO production in LPS-stimulated mammary gland was significantly inhibited in the presence of curcumin (71). These findings suggest that suppression by curcumin of iNOS activity in the mammary glands of irradiated rats helps to prevent radiation-induced tumor initiation. No detectable concentration in serum curcumin was observed in rats fed a diet containing curcumin (69). It is shown that curcumin administered orally is metabolized to tetrahydrocurcumin during absorption through the intestine (72, 73). In fact, tetrahydrocurcumin was detected in the serum of rats fed a diet containing curcumin. Tetrahydrocurcumin has a greater inhibitory effect on the generation of

superoxide anion radicals induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) (74) and lipid peroxidation of erythrocyte membrane ghosts induced by t-butylhydroperoxide than curcumin (75, 76). Oral administration of curcumin does not have any side effects on endocrinologic and physiologic status. These results raise the possibility of clinical application of curcumin in the management of radiodiagnosis to diminish tissue damage caused by irradiation. ANTI-PROMOTION Ovariectomy after irradiation Lactating rats, which have a similar susceptibility to the radiation of developed mammary glands as pregnant rats, have been used to evaluate the role of ovaries in the promotion of tumor development from primordial cells initiated with radiation. Wistar-MS rats were irradiated with 2.6 Gy ␥-rays from a 60Co source for tumor initiation at Day 21 of lactation and were ovariectomized bilaterally 1 month later. During 1 year of observation, a low incidence (4%) of mammary tumorigenesis was found in the ovariectomized rats in comparison with control (35%) parous rats which were irradiated but not ovariectomized (77). When adult

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Table 3. Anti-promotion activity of DHEA, cholesterol-lowering agents, tamoxifen and curcumin on DES-dependent promotion for tumorigenesis from primordial cells initiated by radiation*

Treatment during promotion with DES Control diet DHEA (0.6%, in diet) Control diet Bezafibrate (0.15%, in diet) Control diet Simvastatin (0.03%, in diet) Control pellet Tamoxifen (30%, in pellet) Control diet Curcumin (1%, in diet)

Treatment for initiation 2.6 Gy at Day 20 of pregnancy 2.6 Gy at Day 20 of pregnancy 2.6 Gy at Day 20 of pregnancy 1.5 Gy at Day 21 of lactation 2.6 Gy at Day 20 of pregnancy

Rats with tumors

No. of tumors

No. of rats used

No.

%

AC

FA

No. of tumors per tumor-bearing rat

26 20 48 22 57 22 20 23 39 25

25 7 43 6 50 8 17 1 33 7

96 35 90 27 88 36 85 4 85 28

18 9 27 2 29 2 11 0 19 1

35 6 47 5 56 13 15 1 44 6

2.1 ⫾ 0.4 2.1 ⫾ 1.1 2.0 ⫾ 0.2 1.2 ⫾ 0.2 1.9 ⫾ 0.2 1.9 ⫾ 0.4 1.5 ⫾ 0.3 1.0 1.9 ⫾ 0.2 1.0 ⫾ 0.0

* All rats were treated with DES, as a tumor promoter, for 1 year after irradiation.

female rats of the Sprague-Dawley strain were exposed to fractionated whole-body X-irradiation (1 Gy ⫻ 4 times) and were ovariectomized immediately before the last irradiation, the formation of malignant mammary tumors was completely prevented by ovariectomy (78). From these results, many of the primordial cells in mammary glands that are exposed to radiation do not develop into solid tumors in the absence of ovarian factors such as estrogens or progestins. In other words, substances secreted from the ovary act as promoters of tumorigenesis in irradiated mammary gland.

Dehydroepiandrosterone (DHEA) DHEA, a major secretory C19-steroid of the adrenal cortex, is known to act as a precursor in the biosynthesis of androgens and estrogens. A low plasma level of DHEA may be associated with an increased risk of breast cancer in women (79). In rodents, the oral administration of DHEA inhibits the appearance of spontaneous mammary cancer in female mice (80) and reduces the incidence of NMU-induced and 7,12-dimethylbenz(a)anthracene (DMBA)-induced mammary cancer in rats (81, 82). As shown in Table 3, when pregnant rats received whole body irradiation with 2.6 Gy ␥-rays at Day 20 of pregnancy, and were then treated with DES for a period of 1 year while being fed a diet containing 0.6% DHEA, the incidence (35%) of mammary tumors decreases significantly compared with the incidence (96%) in the control rats (83). The first appearance of palpable tumors in DHEA-fed rats was 4.5 months later than that in the control rats. The concentration of estradiol exceeded, by approximately 6-fold, that in the control rats, and the level of progesterone was decreased by 30%. Interestingly, DHEA prevents DES-induced hypertrophy of pituitary glands and reduces the serum concentration of prolactin. Reduced concentrations of prolactin in pituitary

glands in DHEA-fed rats were detected by immunohistochemical studies, but stimulated the development of mammary glands more than in control rats treated with DES alone. DHEA is metabolized mainly to androst-5-ene3␤,17␤-diol by 17␤-hydroxysteroid dehydrogenase in the mammary glands in rats (84, 85). Because DHEA itself has an extremely low relative binding affinity for estrogen receptor (86), binding of the metabolite to estrogen receptor is well known (87). This observation is consistent with the possibility that androst-5-ene-3␤, 17␤-diol derived from dietary DHEA competes with DES for high-affinity intracellular binding sites for estrogen, thus reducing the promotion activity of DES. Bezafibrate Results from epidemiologic studies have shown that the dietary intake of fatty acids might influence the risk of breast cancer (88, 89). A positive association between dietary fatty acids and tumor growth has also been identified in animal models of mammary carcinogenesis (90). In comparison with animals fed a low-fat diet, female rats fed high-fat diets during the promotional period develop significantly more MNU-induced mammary adenocarcinomas (91, 92). Bezafibrate is an analogue of clofibrate that is currently used in the treatment of hyperlipidemia (93). The treatment of rats with bezafibrate leads to a marked hypolipidemic effect, which is reflected in the reduction of plasma cholesterol and triglyceride levels (94, 95). Pregnant rats were irradiated with 2.6 Gy of ␥-rays at Day 20 of pregnancy, were fed a diet containing 0.15% bezafibrate beginning immediately after weaning, and then were implanted with a DES pellet. Administration of dietary bezafibrate together with DES implantation continued for a period of 1 year, at which time the experiment was terminated. As shown in Table 3, the incidence (27%) of mammary tumors in the bezafibrate-fed rats is less than one-third of that (90%) in the control rats (96). Compared with the control group, the number of mammary tumors per tumor-bearing rat in the

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bezafibrate-treated group was reduced and the appearance of the first palpable tumors was delayed by approximately 5 months in the bezafibrate-fed group. Bezafibrate-fed rats showed a significant decrease in serum prolactin and triglyceride concentrations, and an increase in serum concentrations of estradiol and progesterone in comparison with the control rats. The significant suppression of serum prolactin concentrations in the bezafibrate-fed rats strongly suggests that the mechanism of the chemoprevention by bezafibrate in rats is an alteration in prolactin-induced mammary tumorigenesis. Simvastatin Simvastatin, a synthetic derivative of lovastatin isolated from the culture filtrate of Aspergillus terreus (97), is a prodrug of a specific inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A(HMG-CoA) reductase (98). This compound is lactone. The dihydroxy acid form converted from simvastatin markedly inhibits the activity of HMG-CoA reductase. Mevalonate, produced by HMG-CoA reductase, is also a precursor for the formation of isoprenoid. Transfer of a farnesyl group from farnesyl pyrophosphate to p21ras protein is a universal posttranslation modification among the cellular ras proteins. Inhibition of HMG-CoA reductase leads to the blocking of p21ras farnesylation, thereby reducing its membrane localization (99, 100). Simvastatin prevented DNA replication and cell proliferation in the human breast cancer cell line MCF-7, which activated ras oncogene expression (101). Pregnant rats received whole body irradiation with 2.6 Gy ␥-rays at Day 20 of pregnancy. The irradiated rats were fed a diet containing 0.03% simvastatin immediately after weaning and received a pellet of DES, as tumor promoter, at 30 days after weaning. As shown in Table 3, the administration of dietary simvastatin for 1 year significantly decreased the incidence (36%) of radiation-initiated mammary tumorigenesis, compared with that (88%) of the control rats (102). Also, the development of adenocarcinoma in the simvastatin-fed rats was significantly reduced compared to that in the control rats. After the administration of simvastatin to the irradiated rats, the serum concentration of estradiol was markedly reduced to 20% of that observed in rats fed the control diet, but that of progesterone and of prolactin was not. No significant difference in prolactin cell number or in the expression of prolactin in the pituitary glands of simvastatin-fed rats was observed by immunohistochemical methods. On the other hand, the serum concentration of cholesterol was not reduced by long-term administration of simvastatin in this study. In rats, the activity of HMG-CoA reductase was markedly inhibited by a single administration of simvastatin (103), but the induction of the enzyme by the continuous administration of simvastatin resulted in an increase in the reductase mRNA level (104) and an enhancement of enzyme stability (105). Therefore, it is unlikely that one of the mechanisms behind the chemoprevention of mammary tumors by long-term treatment with simvastatin

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is suppression of the farnesylation of p21ras proteins. Many of the mammary tumors, which developed in the rats fed the control diet, were of type ER⫹PgR⫹, but simvastatin-fed rats showed a significantly decreased incidence of tumors of this type. The maximum number of binding sites for estrogen receptor (ER) and progesterone receptor (PgR) in both adenocarcinoma and fibroadenoma was reduced by simvastatin feeding. The results of the receptor analysis revealed that simvastatin somehow modulates the hormone dependency of the radiation-initiated mammary tumors. Simvastatin is able to induce G1 arrest of ER⫹ mammary tumors cells in the absence of estrogen (106). Because simvastatin markedly reduces the serum concentration of estradiol, an inhibition of the proliferation of ER⫹ tumor cells may have occurred.

Tamoxifen Tamoxifen is the most widely used drug for the treatment of ER-positive breast cancers, and is also under investigation as a chemopreventive agent in healthy women who are at high risk of breast cancer because of their family history (107). The incidence of mammary adenocarcinoma in rats exposed to 4 Gy ␥-rays (108) can be partially suppressed by the administration of tamoxifen for 60 days, i.e., 30 days before and 30 days after irradiation, or for 60 days beginning at 30 days after irradiation. These observations were confirmed by Lemon et al. (109), who reported that longterm treatment with tamoxifen after irradiation also reduced considerably the incidence of mammary carcinoma from 83% in the control to 14%. As shown in Table 3, the incidence (85%) for mammary tumors in rats irradiated with 1.5 Gy ␥-rays at Day 21 of lactation and then administered with DES as a promoter was decreased to 4% with the addition of tamoxifen to DES pellets (39). Thus, tamoxifen is recognized to have antiinitiation and anti-promotion activities in radiation-initiated mammary tumorigenesis. Treatment with DES pellets combined with tamoxifen causes a significant reduction in the concentration of prolactin compared with that in rats implanted with DES pellets as a tumor promoter. Lieberman et al. (110, 111) have reported that the inhibition of estrogenstimulated prolactin synthesis by tamoxifen is caused by a competitive interaction between estrogen and ER in pituitary cells. Tamoxifen treatment is consistently more effective in suppressing mammary tumorigenesis in mice than early ovariectomy (112). The main preventive effect of tamoxifen against DES-promoted mammary tumorigenesis initiated with radiation appears to be the competitive inhibition of the binding of DES to ER in the mammary glands or pituitary gland, or in both. Also, we have completely prevented the formation of radiation-induced mammary tumors in rats by a combination treatment, which is administered with WR2721 before the irradiation and then with tamoxifen during the period of DES-dependent promotion.

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Fig. 2. Chemical prevention of radiation-induced mammary tumorigenesis. ROS ⫽ reactive oxygen species; LPO ⫽ lipid peroxidation; DES ⫽ diethylstilbestrol.

Curcumin DES is a potent tumor promoter of radiation-initiated mammary tumorigenesis (5, 113). An increase in peroxidase activity in the mammary glands is observed to accompany sexual maturity (114). Roy et al. have reported that oxidation of DES by the peroxidase activity of cytochrome P-4501A1 produces some reactive intermediates, presumably the semiquinone and quinone of DES (115). Curcumin is a potent inhibitor of cytochrome P-4501A1 (116). Superoxide anion radicals are generated by redox cycling between DES and its quinone (117). Pregnant rats received whole body irradiation with 2.6 Gy ␥-rays at Day 20 of pregnancy, were fed a diet containing 1% curcumin immediately after weaning, and received DES as a tumor promoter. As shown in Table 3, the administration of dietary curcumin significantly reduced the incidence (28%) of mammary tumors in comparison with that (85%) of rats fed the control diet (118). Also, the incidence (12%) of pituitary tumors in curcumin-fed rats was about one-fifth of that (57%) in the control group. Rats fed the curcumin diet showed a reduced incidence of mammary adenocarcinoma and of ER⫹PgR⫹ tumors, and multiplicity. The serum concentrations of estradiol and prolactin in the curcumin-fed rats were 52% and 80% less than those of control rats, respectively. Furthermore, the serum concentrations of arachidonic acid and eicosapentaenoic acid during ingestion of the curcumin diet were higher than those in rats fed the control diet. Eicosapentaenoic acid is an ␻-3 polyunsaturated fatty acid (PUFA). Growth of a human breast cancer cell line (MDA-MB231) (119) and of a rat mammary adenocarcinoma (120)

was suppressed by ␻-3 PUFA. Dietary ␻-3 PUFA reduces the activity and level of both HMG-CoA reductase (121) and COX-2 (122) in rat mammary glands. Inhibition of HMG-CoA reductase is known to suppress a posttranslational processing of p21ras (123). COX-2 is usually not expressed in most organs, but its expression in certain cells can be rapidly induced by mitogens and hormones (124). Intake of the COX-2 inhibitor nabumetone during the time corresponding to the postinitiation phase has a chemopreventive effect on MNU-induced mammary carcinogenesis in rats (125). In addition, reduced expression of COX-2 in the mammary glands of rats fed ␻-3 PUFA is accompanied by a decreased level of p21ras protein (122). Therefore, it is likely that prevention of mammary carcinogenesis occurs in curcuminfed rats having increased concentrations of serum ␻-3 PUFA. Also, dietary ␻-3 PUFA reduces the activity of NOS in LPS-stimulated macrophage cells (126). The role of free radicals in the tumor promotion stage has been reviewed (127, 128). Considering the above findings, curcumin may scavenge the free radicals derived from the metabolism of DES and exhibit anti-promotion activity. Our observations support the hypothesis that one aspect of the antitumor activity of curcumin during the promotion stage may be also linked to reduction of free radicals, the same as the anti-initiation mechanism. CONCLUSION AND FUTURE PERSPECTIVES In conclusion, radiation-induced mammary tumorigenesis is markedly prevented by administration of radical scav-

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engers or NOS inhibitors during the initiation period. Also, agents for hyperlipidemia or hypercholesterolemia and antiestrogen cause a decrease in the DES-dependent promotion of radiation-induced tumorigenesis (Fig. 2). The development of tumors from mammary primordial cells exposed to radiation is inhibited by oral administration of curcumin,

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which is a major component of turmeric, during the initiation stage or promotion stage. Curcumin does not have any side effects on endocrinologic or physiologic status. These results raise the possibility of clinical application of curcumin in the management of radiodiagnosis to diminish tissue damage caused by radiation.

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