Chapter 19
Chemopreventive Effects of Dibenzoylmethane on Mammary Tumorigenesis 1
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Chuan-Chuan Lin , Chi-Tang Ho , and Mou-Tuan Huang 1
Department of Food Science, China Institute of Technology, Taipei 115, Taiwan Department of Food Science, Rutgers, The State University of New Jersey, 65 Dudley Road, New Brunswick, NJ 08901-8520 laboratory for Cancer Research, School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854-8020
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Dibenzoylmethane (DBM), aβ-diketonestructural analogue of curcumin, has been reported to exhibit chemopreventive activities in mammary tumorigenesis during the past few years. The underlying mechanisms might be complex and have not been well characterized, especially its function at the molecular level. In this report, we overview the recent mechanistic studies of DBM on the multiple stages of mouse mammary carcinogenesis from both chemical and biological aspects.
© 2008 American Chemical Society
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The Chemistry and Biology of Dibenzoylmethane Dibenzoylmethane (DBM), a P-diketone structural analogue of curcumin, has been reported to exhibit anti-tumorigenic and chemopreventive activities during the past few years (7-7). Both DBM and its derivatives have been used as sun-screening agents (8). In biological aspects, DBM inhibits the mutagenicity and nucleic acid binding of chemical carcinogens in vitro (9-11). It modulates the Phase I/Phase II metabolic systems, induces apoptosis in various cancer cells, and, as a metal-chelator, exerts beneficial effects for the ischemic diseases (1,3,5,12). Talalay's group indicated that DBM and its structural derivatives are potent inducers of Phase 2 detoxification enzymes (5). DBM shares a similar structural feature with another naturally-occurring dibenzoylmethane derivative, licodione, [ 1 -(2,4-dihydroxyphenyl)-3-(4hydroxyphenyl)-1,3-propanedione], a biosynthetic intermediate isolated from cultured Glycyrrhiza echinata L. cells (one licorice species) (73). Several isoprenoid-substituted dibenzoylmethanes were isolated from different licorice species (14, 15). Licorice, a sweet-tasting Glycyrrhiza (leguminosae) root, has long been used as a flavoring agent and recently as an anti-ulcer, anti inflammatory agent in Eastern and Western countries (16-18). In addition to the main saponin triterpene constituent, glycyrrhizin, several bioactive flavonoid components, e.g. retrochalcones and dibenzoylmethane derivatives, have been isolated and tested for their biological activities (16). Among them, the licochalcone A from Xin-Jiang licorice showed potent inhibitory effect on DMBA/TPA-induced tumorigenesis (77). Due to the antitumor and anti inflammatory effects of both curcumin and licochalcone, the structural analogues dibenzoylmethane and its derivatives have received more attention, especially its potential use as a chemopreventive agent (6, 7). The studies of the chemopreventive effects of DBM both in vivo and in vitro during the past few years are summarized in Table I and Table II.
The Multiple Stages of Mouse Mammary Carcinogenesis In both rat and mouse models, DBM has been reported to have inhibitory effects toward carcinogen-induced mammary tumorigenesis (2, 6, 7). The typical animal model used for the induction of formation of mouse mammary tumors is shown in Figure 1. The mechanisms involved in this carcinogenic process are also proposed. 7,12-dimethylbenz[a]anthracene (DMBA) is treated orally for 5 weeks and the formation of mammary tumor would occur after the following few weeks. In the initiation stage, the DNA mutation is induced by the bioactivated metabolite of DMBA; in the promotion stage, estrogen is believed to act as an
283 endogenous promoter in this case because estrogen can bind to the estrogenresponsible receptor for induction of cell proliferation. The chemopreventive agents would exert effects on the modulation of both DMBA and estrogen metabolisms or they might act as antiestrogenic agents in this anti-carcinogenic process.
Inhibitory Effect of Dietary DBM on Mammary Tumorigenesis Studies from Huang's laboratory indicated that while dietary curcumin had little or no effect on DMBA-induced breast tumorigenesis in mice, dietary DBM
Table I. Chemopreventive Effects of D B M in vivo Biological activity Inhibit DMBA-induced mouse and rat mammary tumorigenesis Inhibit lymphomas/leukemias in Senear mice Inhibit mammary DMBA-DNA adducts formation in mice and Rats Increase Phase II enzymatic activity in rat liver Inhibit proliferation of mammary gland in mice Decrease several biomarkers related to fat and lower serum estrogen in mice
Reference 6, 7 6 2, 7 4 2 2
Table II. Chemopreventive Effects of D B M in vitro Biological activity Induce Phase 2 detoxification enzymes in murine hepatoma cells Inhibition of carcinogen-DNA adduct formation in MCF-10F Modulate AhR function and expression of cytochromes P450 1A1,1A2, andlBl inHepG2 Inhibit DMBA metabolism and the formation of DMBA-DNA adducts Competitive binding to estrogen receptors with [%]-estradiol Induce HIF-1 alpha and increases expression of VEGF Induce cell cycle deregulation in various human cancer cells
Reference 5 4 3 2 2 12 /
284 Model DMBA Induction (Week 1-5)
Tumor formation * (Week 8-20)
Mechanism Initiation by DMBA
* Promotion by estrogen
effector on DMBA
antiestrogenic activity
m e t a b 0 , l s m
Modulate estrogen metabolism
Figure 1. Typical mouse modelfor DMBA-induced mammary tumorigenesis and possible ways of inhibition
exhibited remarkable inhibitory effects on DMBA-induced mammary tumorigenesis in Senear mice (6). Oral administration of 1 mg of DMBA to female Senear mice (6 weeks old) once a week for 5 weeks, 68% of mice developed mammary tumors (average 1.08 tumors per mouse) at 20 weeks after thefirstdose of DMBA. Feeding 1% DBM in the diet at 2 weeks before DMBA treatment until the end of the experiment, inhibited both the multiplicity and incidence of mammary tumors by 97%. It was of interest to find that 2% curcumin diet had no effect on the DMBA-induced mammary tumor formation in mice. Similar results from another group also showed that DBM, but not curcumin, when added to diets fed to female rat, inhibited the formation of DMBA-DNA adducts in vivo and DMBA-induced rat mammary tumorigenesis (7). In another animal study, we try to examine the inhibitory effect of dietary DBM on cell proliferation of mammary gland and uterus in Senear mice. Immature female Senear mice (26 days of age) were fed with 1% DBM in the diet until the first estrous phase of the estrous cycle. The average bromodeoxyuridine labeling index in the uterus (including epithelium and stroma) and mammary gland were decreased by 40% and 53%, respectively, compared to the corresponding control diet mice (2). This result showed a primary indication of possible antiestrogenic activity of DBM in Senear mice. This report summarizes recent mechanistic studies of DBM on the multiple stages of mouse mammary carcinogenesis from chemical and biological aspects (30, 31).
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The Metabolic Fate of DBM and its Implication on DMBAinduced Mouse Tumorigenesis DMBA, an effective carcinogenic initiator, is metabolically activated by cytochrome P450 oxidase to electrophilic diol-epoxide intermediates, which subsequently interact with DNA to form DMBA-DNA adducts (79, 20). In our previous in vivo and in vitro studies, DBM inhibited DMBA metabolism and formation of DMBA-DNA adducts in a dose-dependent manner (2, 27). Investigation of the underlying mechanism regarding the involvement of Pdiketone moiety of DBM on mammary tumorigenesis is important. The Pdiketone functionality in curcumin has been shown to exhibit antioxidative activity on tert-butylhydroperoxide-induced lipid peroxidation of erythrocyte membrane ghosts (22). Talalay also reported the potency of DBM as an inducer of Phase II detoxification enzymes, in part due to the P-diketone functionality (5). However, not any report published so far has concerned the influence of the p-diketone group on cytochrome P450 Phase I metabolizing enzymes, since DMBA needs to be oxidatively metabolized to bioactive carcinogen (79). We examined the metabolic fate of DBM by NADPH-dependent cytochrome P450 enzymes in mouse liver microsomes. The identification of a major reductive DBM metabolite as well as several minor metabolites in pdiketone moiety from incubation with mouse liver microsomes in vitro is presented. Meanwhile, the possible metabolic pathway of DBM is proposed in Figure 2. These may provide partially the explanation from chemical aspects of the role of DBM as a modulator of the cytochrome P450 reductase that is required for the function of oxidase to metabolize DMBA. This might also result in the inhibition of DBM on DMBA-induced tumorigenesis.
The Inhibitory Effect of DBM on Estradiol-induced Mammary Proliferation Overexpressions of oncogenes induced by estradiol (E ) have been suggested to lead to the mammary tumorigenesis in animal and proliferation of human cancerous cells (23-26). Specifically, the synergistic effects of oncogenic expressions, e.g., c-myc, ras, bcl-2, and telomerase were observed in both transgenic mice and cultured cells, resulting in abrupt cellular proliferation and tumor formation (27-29). Consistently, estrogen response elements (EREs), which are required for gene expressions, have been identified in either promoter positions or coding sequences in several oncogenes (23-26). 2
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Bezopic acid Dihydmchakane
Figure 2. Proposed metabolic pathway of DBM by mouse liver microsomes
To examine how DBM affects E -dependent cell proliferation, the expressions of four oncogenes, bcl-2, c-myc, Ha-ras and hTERT, with their EREs having been identified in estrogen receptor-positive MCF-7 cells, were examined by quantitative RT-PCR, a technique for quantitative analysis of gene expression with high specificity. In a time-course study, MCF-7 cells were treated with 100 nM of E for several time intervals and the results indicated that E -induced c-myc, Ha-ras and bcl-2 reached their maximum expression levels after induction for 2 hrs, whereas hTERT required 8 hrs to aggrandize its climax (data not shown). As shown in Figure 3A, the expression levels of hTERT, cmyc, Ha-ras and bcl-2 in E -treated cells were increased by 4.6,4.1,2.4, and 5.4 fold, respectively, compared to the E -untreated control. Treatment of 10 j i M of DBM together with 100 nM E reduced the expression of these four oncogenes to the basal levels, for c-myc, where a lesser extent of attenuation was observed. The inhibitory pattern of DBM was also compared to that of tamoxifen, a SERM that antagonizes the estrogenic action of E in MCF-7 cells. Both DBM and 2
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287 tamoxifen exhibited similar patterns of inhibition as shown in Figure 3. Tamoxifen decreased the expressions of hTERT, c-myc, and bcl-2. However, tamoxifen did not show significant reduction of expression in Ha-ras. These results suggest that DBM inhibits the expression of E -regulated oncogenes, which might attribute to its inhibitory effect on E -stimulated cellular proliferation. 2
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Figure 3. DBM and tamoxifen (Tarn) affected the expressions of the E2-ERERE-dependent oncogenes in MCF-7 cells. MCF-7 cells were treated with EtOH 100nME2, 100 nME2+10 IJMDBM, and 10 fjMDBM (A) or EtOH, 100 nME2 100 nME2+l \iM Tarn, and 1 \lM Tarn (B)for 8 hrs to detect hTERT expression or 2 hrs to others. RNA was extracted and cDNA was then synthesized according to the standard protocol. The expression of hTERT, cmyCy Ha-ras and bcl-2 were measured by quantitative PCR analysis. Data are presented as the mean ± S.D.from triplicate determinations. Significant differences (P
