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Inhibition of Benzo(a)pyrene Diol-Epoxide-induced Transactivation of Activated Protein 1 and Nuclear Factor B by Black Raspberry Extracts¹

Nelson Institute of Environmental Medicine, New York University School of Medicine, Tuxedo, New York 10987 [C. H., Y. H., J. L., W. H., M-s. T.], and Division of Environmental Health Sciences, School of Public Health [R. A., G. D. S.] and College of Pharmacy [N. S., J. C.], The Ohio State University, Columbus, Ohio 43210

	ABSTRACT

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Freeze-dried black raspberries have been shown to inhibit the development of chemically induced esophageal and colon cancer in rodents.In addition, organic extracts of black raspberries inhibit benzo(a)pyrene (BaP)-induced cell transformation in vitro. The molecular mechanisms through which black raspberries inhibit carcinogenesis remain unclear. We investigated the effects of black raspberry extracts on transactivation of activated protein 1 (AP-1) and nuclear factor B (NFB) induced by BaP diol-epoxide (BPDE), the ultimate carcinogen of BaP, in mouse epidermal JB6 Cl 41 (Cl 41) cells. Black raspberries were extracted with methanol, and the methanol extract was partitioned and chromatographed into several fractions designated RU-F003, RU-F004, RU-DM, and RU-ME. Pretreatment of Cl 41 cells with RU-F003, RU-DM, or RU-ME resulted in an inhibition of BPDE-induced AP-1 and NFB activities. The RU-ME fraction was the most potent inhibitor among the fractions tested. In contrast, fraction RU-F004 did not inhibit BPDE-induced AP-1 or NFB activities in Cl 41 cells. The inhibitory effects of RU-ME on BPDE-induced activation of AP-1 and NFB appear to be mediated via inhibition of mitogen activated protein kinase activation and inhibitory subunit B phosphorylation, respectively. Pretreatment of cells with berry fractions did not result in an inhibition of BPDE binding to DNA; thus, this was not a mechanism of reduced AP-1 and NFB activities. None of the fractions was found to affect p53-dependent transcription activity. In view of the important roles of AP-1 and NFB in tumor promotion/progression, these results suggest that the ability of black raspberries to inhibit tumor development may be mediated by impairing signal transduction pathways leading to activation of AP-1 and NFB. The RU-ME fraction appears to be the major fraction responsible for the inhibitory activity of black raspberries.

	INTRODUCTION

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Epidemiological data suggest an inverse relationship between consumption of fruit and vegetables and the occurrence of several types of cancer in humans (1 , 2) . On the basis of these data, it is estimated that if all citizens consumed at least five helpings of fruits and vegetables per day, the overall occurrence of cancer in the United States could be reduced at least 20–30% (2) . In addition, experiments in animal models have provided direct evidence of the protective effects of fruits and vegetables on tumor development. Dietary supplementation with various freeze-dried vegetables was shown recently to significantly reduce azoxymethane-induced aberrant crypt foci, a surrogate end point biomarker for tumor development, in the rat colon (3) . Studies have also demonstrated the protective effects of tomato juice, paprika juice, garlic, soybeans, and dry beans on tumor development in various animal model systems when added to the water supply or mixed in the diet (4, 5, 6, 7, 8, 9) . We have applied a similar food-based approach to the prevention of cancer in a rat model of esophageal squamous cell carcinoma (10, 11, 12) . We have reported recently on the ability of dietary freeze-dried strawberries (10 , 11) and black raspberries (12) to inhibit NMBA⁴ tumorigenesis in the rat esophagus. Both berry types produced an approximate 40–60% reduction in esophageal tumor multiplicity when administered at dietary concentrations of 5% and 10% before, during, and after treatment of the rats with NMBA (10, 11, 12) . In addition, pretreatment of rats with dietary berries was shown to reduce the formation of O⁶ -methylguanine adducts from NMBA in esophageal DNA, indicating that the berries influenced the metabolism of the carcinogen (10, 11, 12) .

When administered in the diet after treatment of the rats with NMBA, BRB [Rosaceae (Family); Rubus (Genus); occidentalis (species)] inhibited the esophageal tumor response to NMBA by approximately 30–50% (12) . The berries were found to be capable of suppressing the development of preneoplastic lesions (hyperplasia, and low- and high-grade dysplasia) into papillomas. Immunohistochemical staining for proliferating cell nuclear antigen indicated that the berries reduced the rate of proliferation of preneoplastic cells in NMBA-treated esophagi essentially to levels seen in control animals. In addition, we have shown recently that BRB produced approximately 30–80% inhibition of azoxymethane-induced tumors in the rat colon when administered postinitiation at dietary concentrations of 2.5–10% (13) . Finally, we also found that one fraction (RU-ME) of a methanol extract of black raspberries inhibited BaP-induced cell transformation in a dose-dependent manner (14) . Recent data in our laboratory indicate that coincubation of JB6 Cl 41 cells with the RU-ME fraction of black raspberries results in inhibition of TPA- and epidermal growth factor-induced cell transformation.⁵ Although the specific inhibitory components in the berries have not as yet been identified, our studies have revealed that berries contain multiple compounds with known chemopreventive activity including vitamin A, vitamin C, vitamin E, folic acid, calcium, selenium, ß-carotene, -carotene, ellagic acid, ferulic acid, coumaric acid, and multiple anthocyanins and phytosterols (11 , 14) .

Activation of the MAPKs/AP-1 pathways has been shown to be involved in human colon cancer development (15 , 16) . Similarly, we have found recently that c-jun, a major component of AP-1, is increased in esophageal tissues after treatment of rats with NMBA,⁶ suggesting that AP-1 may also be involved in esophageal carcinogenesis. In view of the essential roles of AP-1 and NFB in tumor promotion (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36) , we investigated the potential effect of black raspberry extracts on AP-1 transactivation in the present study. Mouse epidermal JB6 Cl 41 cells were used in the study because they are well characterized and widely used for studies of the role of signal transduction pathways in tumor promotion (17, 18, 19, 20, 21, 22) . In addition, we have stably transfected these cells with AP-1- and NFB-luciferase reporter for AP-1 and NFB activities, and have shown recently that these genes are inducible with BPDE, the ultimate carcinogen of BaP. Finally, JB6 cells are derived from a squamous epithelium similar to the esophagus, thus it seemed logical to use them for study pending the availability of esophageal cell cultures. Data from our studies suggest that the down-regulation of AP-1 and NFB in JB6 Cl 41 cells resides most significantly in one organic fraction of black raspberries.

	MATERIALS AND METHODS

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Cells and Culture Conditions.
Mouse epidermal cell line, JB6 Cl 41 (Cl 41), and three cell lines derived from Cl 41 cells were used in these studies. These lines were transfected stably with either an AP-1-luciferase reporter (P⁺1–1 cells), a NFB-luciferase reporter (Cl 41 NFB mass1 cells), or a p53-luciferase reporter (Cl 41 PG13 mass1 cells; Refs. 23, 24, 25 ). All four of the cell lines; i.e., Cl 41, P⁺1–1, Cl 41 NFB mass1, and Cl 41 PG13 mass1, were cultured in Eagle’s MEM supplemented with 5% FBS, 2 mM L-glutamine, and 25 µg of gentamicin/ml. Eagle’s MEM was purchased from Calbiochem (San Diego, CA), and L-glutamine, gentamicin, and FBS from Life Technologies, Inc. (Rockville, MD). The cells were cultured at 37°C in a humidified atmosphere of 5% CO₂ in air. The cultures were dissociated with trypsin and transferred to new 75-cm² culture flasks (Fisher, Pittsburgh, PA) from one to three times per week.

Black Raspberries and Extracts.
Fig. 1 presents a schematic for the preparation of the black raspberry extracts used in this study. Ripe black raspberries (RU) were washed immediately after picking, frozen at -20°C, then freeze-dried as described by Stoner et al. (10) . Approximately one pound of freeze-dried berries was extracted in 3 volumes of methanol overnight for 3 nights. The extract was filtered and then dried under vacuum at 60°C to produce Fraction RU-F001. The residue from fraction RU-F001 (RU-F002) represented solid residue that was not additionally processed. A portion of RU-F001 was partitioned with water:dichloromethane (1:1). The aqueous layer was concentrated under vacuum and dried (RU-F003). The organic (dichloromethane) layer was vacuumed dried at 60°C resulting in a water-insoluble fraction (RU-F004). A small amount of insoluble fraction, RU-F005, was obtained from the interface between the aqueous and organic layer. Additional RU-F001 was dissolved in methanol and allowed to evaporate. The resulting precipitate was chromatographed on a silica gel column and eluted by dichloromethane:methanol (1:1). The resulting nonpolar eluate (RU-DM) and polar fraction (RU-ME) were obtained. All of the extracts were stored at -20°C in the dark until examined in the assays. For cell treatment, each extract was dissolved in DMSO to give a final concentration of 50 mg/ml of extract and frozen at -70°C for use in the assays.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Procedure for preparation of BRB fractions.

AP-1 Activity Assay.
Confluent monolayers of P⁺1–1 cells were trypsinized, and 8 x 10³ viable cells suspended in 100 µl of MEM supplemented with 5% FBS were added to each well of 96-well plates. Plates were incubated at 37°C in a humidified atmosphere of 5% CO₂ in air. After the cell density reached 80–90%, the culture medium was replaced with an equal volume of MEM supplemented with 0.1% FBS and 2 mM L-glutamine. Twelve h later, the cells were treated with different fractions of black raspberries dissolved in DMSO for 30 min at concentrations ranging from 1 to 100 µg/ml. Cells were then exposed to BPDE at a final concentration of 2 µM. The cells were extracted with lysis buffer (Promega) at various periods of time (6–48 h) as indicated in the figure legends after BPDE exposure, and the luciferase activity was determined by the luciferase assay using a luminometer (Wallac 1420 Victor 2 multilable counter system) after the addition of 100 µl of lysis buffer for 30 min at 40° C. The results are expressed as AP-1 activity relative to control medium containing DMSO (0.1% v/v) only (relative AP-1 activity).

NFB and p53-dependent Transcription Activity Assays.
The same procedure as described above for measuring the effects of berry fractions on BPDE-induced AP-1 activity in P⁺1–1 cells was used for determining the effects of the same berry fractions on BPDE-induced NFB activity in NFB mass1 cells, and p53-dependent transcription activity in PG-13 mass1 cells. The results were expressed as either NFB activity or p53-dependent transcription activity relative to control medium containing DMSO.

BPDE-DNA Adduct Assay.
The effects of one berry fraction; i.e., RU-ME, on the formation of BPDE-DNA adducts in cultured Cl 41 cells was determined. A stock solution of [ ³H]BPDE (specific activity, 2210 mCi/mmol) was freshly prepared in tetrahydrofuran/triethylamine (19:1). C1 41 cells grown to 50–70% confluency in serum-free medium were washed three times with DPBS and treated with either a mixture of [ ³H]BPDE (2 µM) and berry fraction RU-ME (50 µg/ml), or [ ³H]BPDE (2 µM) alone. The mixture was kept at room temperature for 15 min in the dark room and then added to the C1 41 cells. Cells were incubated with the mixture or with [ ³H]BPDE alone, at 37°C for 1 h in the dark, and then washed three times with DPBS to remove unbound [ ³H]BPDE. The cells were then incubated in lysis buffer [0.5% SDS, 10 mM Tris (pH 7.8), 10 mM EDTA, 10 mM NaCl, and 100 µg/ml proteinase K] for 1 h at room temperature. Genomic DNA was extracted with phenol and diethyl ether, then precipitated in 2.5 volumes of ethanol and resuspended in TE buffer [10 mM Tris (pH 7.5) and 1 mM EDTA]. RNA was removed by treatment with RNase A (50 µg/ml) followed by phenol and diethyl ether extractions, and ethanol precipitation. The purified genomic DNA was quantitated by UV spectrophotometry, and the quality of the DNA was checked by electrophoresis in 0.5% agarose gels, as well as by measurement of the A₂₆₀:A₂₈₀ ratio. To determine the extent of [³H]BPDE-DNA adduct formation in the genomic DNA, a known quantity of DNA samples was mixed with liquid scintillation counter mixture (Fisher Scientific Co., Pittsburgh, PA), and the [³H] cpm was counted in a 1219 RACKBETA scintillation counter (LKB Wallac, Dayton, OH). The number of BPDE-induced DNA adducts in 10-kb genomic DNA was calculated based on the specific activity of the [³H]BPDE after correction for counting efficiency.

Kinase Phosphorylation Assay.
Cl 41 cells were cultured in monolayers in six-well plates. After the cell density reached 70–80%, the serum in the medium was reduced from 5% to 0.1% and cultured for 40–45 h. Cells were incubated in MEM containing 0.1% serum and 2 mM L-glutamine for 3–4 h at 37°C. Cells were then incubated with BPDE (2 µM) for different periods of time and extracted with Tris-glycine SDS sample buffer (Invitrogen, Carlsbad, CA). Western blots were performed with either phosphospecific antibodies or nonphosphorylated antibodies against various kinases, including ERKs, JNKs, and p38 kinase, and also against IB. The protein band specifically bound to the primary antibody was detected using an antirabbit IgG-AP-linked and an ECF Western blotting system (Amersham Bioscience, Piscataway, NJ).

Statistical Analysis.
The Student t test was used to determine the significance of differences between both AP-1 and NFB activities in cells treated with berry fraction + BPDE, BPDE alone, or DMSO alone. The differences were considered significant at a P of <0.05.

	RESULTS

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Effect of Berry Fractions on BPDE-induced Activation of AP-1 in P⁺1–1 1 Cells.
To investigate the potential effects of BRB fractions on BPDE-induced AP-1 activation, P⁺1–1 cells were pretreated with each of four BRB fractions (RU-F003, RU-F004, RU-DM, and RU-ME) at 25 µg/ml for 30 min, and then exposed to 2 µM of BPDE to induce AP-1. Pretreatment of P⁺1–1 cells with the RU-F003, RU-DM, or RU-ME fractions resulted in a significant inhibition (P < 0.05) of BPDE-induced AP-1 activity, whereas the RU-F004 extract had no effect (Fig. 2) . The RU-ME fraction was the most potent inhibitor of AP-1 activity among the extracts tested (Fig. 2) , which is consistent with its potency as an inhibitor of BaP-induced cell transformation (14) . The RU-ME and RU-F003 fractions were both inhibitory when added to the medium at only 1 µg/ml (Fig. 3, A and B) . The inhibitory effect of RU-ME on BPDE-induced AP-1 activity was present at all of the time points tested (Fig. 3C) .

View larger version (32K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Inhibition of AP-1 by BRB extract fractions. P+1–1 cells (8 x 103) were seeded into each well of 96-well plates and cultured in 5% FBS MEM at 37°C. The medium was replaced with 0.1% FBS MEM after the cell density reached 80–90%. Twelve h later, cells were pretreated with various BRB extracts for 30 min and then treated with BPDE (2 µM) for induction of AP-1. After 24 h of incubation, the cells were extracted with lysis buffer, and luciferase activity was measured using the Promega luciferase assay as described in "Materials and Methods." The results are presented as AP-1 activity in treated versus untreated wells (23 24 25) . Each bar indicates the mean of four repeat wells; bars, ±SD. * indicates a significant decrease from BPDE-treated cells (P < 0.05).

View larger version (11K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Dose-dependent inhibition of AP-1 activity by RU-ME and RU-F003. P+1–1 cells (8 x 103) were seeded into each well of 96-well plates and cultured as described in Fig. 1 until the cell density reached 80–90%. For the dose-response study, the cells were pretreated with various concentrations of either RU-ME (A) or RU-F003 (B) for 30 min and then treated with BPDE (2 µM) for induction of AP-1. After 24 h of incubation, the cells were extracted with lysis buffer. For the time course study (C), the cells were treated with 20 µg/ml RU-ME for 30 min and then exposed to BPDE (2 µM) for the time points indicated. The cells were extracted with lysis buffer. The luciferase activity was measured as described in "Materials and Methods." The results are presented as AP-1 activity in treated versus untreated cells (23 24 25) . Each bar indicates the mean of four repeat wells; bars, ±SD. * indicates a significant decrease from BPDE alone (P < 0.05).

Effect of Berry Fractions on Induction of NFB by BPDE.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Inhibition of NFB activities by BRB extracts. NFB mass1 cells (8 x 103) were seeded into each well of 96-well plates and cultured as described in Fig. 1 until the cell density reached 80–90%. The cells were pretreated with either (A) various extracts of BRB at 25 µg/ml or (B) various concentrations of RU-ME for 30 min, and then with BPDE (2 µM) to induce NFB. After 24 h of incubation, the cells were extracted with lysis buffer. For the time course study (C), the cells were treated with 20 µg/ml RU-ME for 30 min and then exposed to BPDE (2 µM) for the time points indicated. The cells were extracted with lysis buffer, and luciferase activity was measured as described in "Materials and Methods." The results are presented as NFB activity in treated cultures versus medium controls (25) . Each bar indicates the mean of four repeat wells; bars, ±SD. * indicates a significant decrease from BPDE alone (P < 0.05).

View larger version (38K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Effect of BRB extracts on BPDE-induced p53-dependent transcription activity. PG13 mass1 cells (8 x 103) were seeded into each well of 96-well plates and cultured as described in Fig. 1 . The cells were pretreated with various extracts of BRB for 30 min and then with BPDE (2 µM) for p53 induction. After 24 h of incubation, cells were extracted with lysis buffer, and luciferase activity was measured. The results are presented as p53-dependent transcription activity in treated culture versus medium controls (23) . Each bar indicates the mean of four repeat wells; bars, ±SD.

Pretreatment or Simultaneous Coincubation of Cells with RU-ME Is Required for Inhibition of BPDE-induced Activation of AP-1 and NFB in Cl 41 Cells.

View larger version (17K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. No inhibitory effect of RU-ME on activation of AP-1 (A) and NFB (B) when added 3 h after cells were treated with BPDE. P+1–1 or NFB mass1 cells were seeded into each well of 96-well plates and cultured as described in Fig. 1 . The cells were then treated with RU-ME (50 µg/ml) either at 30 min before BPDE exposure or simultaneously with BPDE, or at 3–6 h after BPDE exposure. The cells were extracted with lysis buffer at 24 h after BPDE treatment, and luciferase activity was measured. The results are presented as AP-1 or NFB activity in treated culture versus medium controls (23 24 25) . Each bar indicates the mean of four repeat wells; bars, ±SD.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 7. Effects of RU-ME on BPDE-induced DNA adduct formation. C1 41 cells at 50–70% confluency were washed with DPBS. The mixture of [3H]BPDE and berry fraction RU-ME, or [3H]BPDE alone, was added to serum-free culture medium as described in "Materials and Methods." The mixture was kept at room temperature for 15 min in the dark, and then added to the C1 41 cells. Genomic DNA was extracted from the cells as described in "Materials and Methods." The extent of DNA adduct formation by [3H]BPDE in the genomic DNA was determined by scintillation counting (A). The number of BPDE-induced DNA adducts in 10-kb genomic DNA was calculated from the specific activity of the [3H]BPDE after correction for counting efficiency (B). * indicates a significant increase from BPDE alone (P < 0.05).

View larger version (57K): [in this window] [in a new window] [Download PPT slide]   Fig. 8. Inhibition of BPDE-induced activation of MAPKs (A), and IB phosphorylation and degradation (B) by RU-ME. Cl 41 cells were seeded into each well of six-well plates and cultured in 5% FBS MEM. The culture medium was replaced with 0.1% FBS MEM after the cell density reached 80–90%. Forty-eight h later, the cells were pretreated with RU-ME for 30 min and then exposed to BPDE (2 µM). Cells were washed once with ice-cold PBS and extracted with SDS sample buffer at either 90 min or 270 min after BPDE treatment. Cell extracts were separated on polyacrylamide-SDS gels, transferred, and probed with either phosphospecific antibodies or nonphosphorylated antibodies against various kinases, including (A) ERKs, JNKs, and p38 kinase, and (B) IB. The protein band-specific binding with primary antibodies was detected by using antirabbit IgG-AP-linked and an ECF Western blotting system (20 , 23 , 25) .

Inhibition of NFB by RU-ME Is Attributable to Its Influence on BPDE-induced Phosphorylation and Degradation of IB.

	DISCUSSION

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Epidemiological studies suggest a protective effect of fruits and vegetables on cancer development in humans (1 , 2) . Experimentally, we have demonstrated a protective effect of BRB on the development of chemically induced esophageal and colon cancer in rodents (12 , 13) . In addition, we reported the ability of organic fractions of black raspberries to inhibit BaP-induced transformation of Syrian hamster embryo cells in vitro (14) . To further understand the molecular mechanisms through which black raspberries inhibit carcinogenesis, we evaluated the effect of black raspberry fractions on BPDE-induced AP-1 and NFB activation in mouse epidermal JB6 Cl 41 cells (Cl 41 cells). Pretreatment of Cl 41 cells with RU-F003, RU-DM, or RU-ME fractions resulted in an inhibition of both AP-1 and NFB activities, whereas they did not affect BPDE-induced increase in p53-dependent transcription activity. The RU-ME extract was found to be the most potent for inhibition of BPDE-induced AP-1 and NFB activities. Studies are currently underway to determine the chemical composition of the RU-ME fraction, and to isolate individual compounds with inhibitory activity toward both AP-1 and NFB.

Transcription factor AP-1 consists of homodimers and heterodimers of Jun (v-Jun, c-Jun, JunB, and JunD), Fos (v-Fos, c-Fos, FosB, Fra1, and Fra2), or activating transcription factor (ATF2, ATF3/LRF1, and B-ATF) proteins (35) . AP-1 and its regulated gene expression have been shown to play an important role in cell proliferation, cell cycle regulation, and tumor promotion (35 , 36) . AP-1 activity was found to be progressively elevated in mouse epidermal JB6 cells representing various stages of tumor promotion (17) . Activation of AP-1 appears to be required for the preneoplastic-to-neoplastic progression of JB6 cells (18, 19, 20, 21, 22) .

NFB consists of two major subunits, p50 and p65 (Rel-A; Refs. 27 , 28 ). It exists in cells as an inactive cytoplasm precursor by forming a complex with IB, an inhibitor for NFB (27 , 28) . The signal leading to IB phosphorylation triggers the release of NFB from IB, resulting in the activation and translocation of NFB from the cytoplasm to the nucleus where NFB binds to the promoter region of its specific targeting genes (27 , 28) . Similar to AP-1, NFB was first considered to be a mediator of tumor promotion because of its ability to alter gene expression in response to tumor promoters and oncogenes, including TPA and tumor necrosis factor , UV radiation, metals, reactive oxygen species, and HER-2/neu (31) . The two members of the NFB family, v-rel and p52/lyt-10, and IB family member Bcl-3 are potentially oncogenic (24) . H-Ras and Raf-1 can also activate NFB in transfection assay (32 , 33) . Overexpression of the NFB inhibitor, IB, blocked the ability of oncogenic Ras alleles to induce focus formation in 3T3 cells (34) .

The present study demonstrates the ability of the RU-F003, RU-DM, and RU-ME fractions of BRB to inhibit the activities of the important transactivator proteins, AP-1 and NFB. Fraction RU-F004 did not exhibit this ability, thus, comparative chemical analysis of this fraction versus the RU-F003, RU-DM, and RU-ME fractions might permit the identification of specific compounds that exhibit inhibitory effects. RU-ME was the most potent fraction for inhibition of BPDE-induced AP-1 and NFB activities. The inhibitory effects of RU-ME on BPDE-induced activation of AP-1 and NFB appears to be mediated via inhibition of MAPK activation and IB phosphorylation, respectively. The amount of BPDE-DNA adduct formation in cells treated with BPDE in the presence of RU-ME was higher than that in cells treated with BPDE alone. These results suggest that compounds in RU-ME do not scavenge BPDE, and the cancer-preventive activity of RU-ME does not involve effects on initiation events. It is known that BPDE is relatively labile in aqueous solution. It is possible that compounds in RU-ME may increase BPDE stability in aqueous medium and/or the uptake of this carcinogen by cells.

The environmental carcinogen BaP is metabolized in vivo in humans and animals to its ultimate carcinogenic form, BPDE (38, 39, 40) . For example, when activated with rat liver microsomes, BaP is almost exclusively (98%) metabolized to BPDE (40) . Mouse skin tumorigenicity studies indicate that BPDE acts as both tumor initiator and promoter (40, 41, 42, 43, 44) . Significant skin tumor initiation by BPDE was observed when a single topical application of BPDE was followed by twice weekly applications of TPA for 24 weeks (45) . Repeated topical application of BPDE on C57BL/6J mouse skin is very effective in producing hyperplasia and, ultimately tumors (43 , 44) . The mutagenic and DNA-damaging effects of BPDE, which are responsible for tumor initiation, are extensively documented. One of the genes in mouse skin that is mutated by BPDE is c-Ha-ras, which plays a role in tumor development (46) . Mechanism(s) underlying the tumor promotion effects of BPDE are not well understood. Our recent studies showed that treatment of Cl 41 cells with BPDE resulted in a marked activation of AP-1 and NFB, whereas BaP only induces a slight activation.⁷ Recent data in our laboratory also suggest that the activation of AP-1 and its upstream MAPKs, including ERKs, JNKs and p38K, by BPDE occurs through phosphatidylinositol 3'-kinase/Akt-dependent and p70^S6k-independent pathways. Because BPDE is capable of inducing oxidative DNA damage, we speculate that BPDE-induced activation of AP-1 and NFB may be mediated by agents that induce oxidative stress. The inhibitory effect of BRB extracts on BPDE-induced AP-1 activation may be because of their ability to reduce oxidative stress. This notion is supported by recent findings that berry extracts scavenge reactive oxygen species generated by metals (data not shown).

In summary, our results suggest that the inhibition of AP-1 and NFB activities by berry extracts is at least partially responsible for their ability to inhibit cell proliferation in vitro. Investigations are under way to determine whether this is the case in vivo, using the rat model of esophageal cancer.

	ACKNOWLEDGMENTS

 
We thank the Cancer Research Program of the National Cancer Institute, Division of Cancer Cause and Prevention, Bethesda, MD, for supplying [³H]BPDE.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported in part by National Cancer Institute Grant CA 46535 and by National Institute of Environmental Health Sciences 5P30 ES00260.

² To whom requests for reprints should be addressed, at Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge Road, Tuxedo, NY 10987. Phone: (845) 731-3519; Fax: (845) 351-2118; E-mail: chuanshu{at}env.med.nyu.edu

³ This author is a summer student from Monroe-Woodbury High School, 155 Dunderberg Road, Central Valley, NY 10917.

⁴ The abbreviations used are: NMBA, N-nitrosomethylbenzylamine; AP-1, activated protein 1; BaP, benzo(a)pyrene; BRB, freeze-dried black raspberries; BPDE, benzo(a)pyrene diol-epoxide; ERK, extracellular signal-regulated protein kinase; FBS, fetal bovine serum; IB, inhibitory subunit -B; IL, interleukin; JNK, c-Jun NH₂-terminal kinase; MAPK, mitogen-activated protein kinase; NFB, nuclear factor B; P⁺, tumor promoter-sensitive; P^-, tumor promoter-resistant; RU, Rubus occidentalis; TPA, 12-O-tetradecanoylphorbol-13-acetate; DPBS, Dulbecco’s PBS.

⁵ Huang, C., Li, J., and Stoner, G., unpublished observations.

⁶ Stoner, G., Habib, S., and Huang, C., unpublished observations.

⁷ Li, C., Tang, M-S., and Huang, C, unpublished observations.

Received 5/28/02. Accepted 9/25/02.

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