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[Cancer Research 62, 6857-6863, December 1, 2002]
© 2002 American Association for Cancer Research

Epidemiology and Prevention

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

Chuanshu Huang2, Yi Huang3, Jingxia Li, Wenwei Hu, Robeena Aziz, Moon-shong Tang, Nanjun Sun, John Cassady and Gary D. Stoner

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
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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 {kappa}B (NF{kappa}B) 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 NF{kappa}B 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 NF{kappa}B activities in Cl 41 cells. The inhibitory effects of RU-ME on BPDE-induced activation of AP-1 and NF{kappa}B appear to be mediated via inhibition of mitogen activated protein kinase activation and inhibitory subunit {kappa}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 NF{kappa}B activities. None of the fractions was found to affect p53-dependent transcription activity. In view of the important roles of AP-1 and NF{kappa}B 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 NF{kappa}B. The RU-ME fraction appears to be the major fraction responsible for the inhibitory activity of black raspberries.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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, {alpha}-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 NF{kappa}B 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 NF{kappa}B-luciferase reporter for AP-1 and NF{kappa}B 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 NF{kappa}B in JB6 Cl 41 cells resides most significantly in one organic fraction of black raspberries.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 NF{kappa}B-luciferase reporter (Cl 41 NF{kappa}B 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 NF{kappa}B 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. 1Citation 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.



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  		Fig. 1. Procedure for preparation of BRB fractions.

 
Other Reagents.
The substrate for the luciferase assay was purchased from Promega (Madison, WI); BPDE was from Sigma (St. Louis, MO); and the phosphospecific antibodies against various phosphorylated sites of ERKs, p38 kinase, JNKs, and I{kappa}B{alpha} were from New England Biolaboratories (Beverly, MA). (±)-r-7, t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydro[1,3-3H]BaP ([3H]BPDE, specific activity; 2210 mCi/mmol) was purchased from ChemSyn Science Laboratories (NCI Chemical Carcinogen Repository, Kansas City, MO).

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).

NF{kappa}B 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 NF{kappa}B activity in NF{kappa}B mass1 cells, and p53-dependent transcription activity in PG-13 mass1 cells. The results were expressed as either NF{kappa}B 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 I{kappa}B{alpha}. 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 NF{kappa}B 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
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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)Citation . The RU-ME fraction was the most potent inhibitor of AP-1 activity among the extracts tested (Fig. 2)Citation , 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)Citation . The inhibitory effect of RU-ME on BPDE-induced AP-1 activity was present at all of the time points tested (Fig. 3C)Citation .



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  		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).

 


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  		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. 1Citation 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 NF{kappa}B by BPDE.
We also examined the effects of the four BRB fractions on BPDE-induced NF{kappa}B activity in NF{kappa}B mass1 cells. Preincubation of the cells with the RU-F003, RU-DM, or the RU-ME fraction led to a significant inhibition (P < 0.05) of BPDE-induced NF{kappa}B activity in the cells (Fig. 4A)Citation . In contrast, fraction RU-F004 did not inhibit NF{kappa}B activity (Fig. 4A)Citation . The RU-ME fraction was the most potent inhibitor of NF{kappa}B activity among the fractions tested (Fig. 4A)Citation . The inhibitory effect of RU-ME on BPDE-induced NF{kappa}B activity appeared to be in dose- and time-dependent manner (Fig. 4, B and C)Citation . Considering the important roles of AP-1 and NF{kappa}B in tumor growth we hypothesize that inhibition of AP-1 and NF{kappa}B are important for the inhibitory effect of BRB on the growth of preneoplastic cells in the rat esophagus in vivo. In contrast, BPDE-induced p53-dependent activation was not affected by any of the fractions tested (Fig. 5)Citation .



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  		Fig. 4. Inhibition of NF{kappa}B activities by BRB extracts. NF{kappa}B mass1 cells (8 x 103) were seeded into each well of 96-well plates and cultured as described in Fig. 1Citation 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 NF{kappa}B. 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 NF{kappa}B 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).

 


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  		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. 1Citation . 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 NF{kappa}B in Cl 41 Cells.
To determine the conditions under which the berry fractions inhibit BPDE-induced activation of AP-1 and NF{kappa}B in Cl 41 cells, we added the most active fraction, RU-ME, to cultured Cl 41 cells at different times before or after exposure of the cells to 2 µM BPDE. The inhibitory effect of the RU-ME fraction on both AP-1 and NF{kappa}B occurred only when RU-ME was added either before or along with the BPDE (Fig. 6)Citation . RU-ME was not effective when added to the cells 3 h after treatment with BPDE (Fig. 6)Citation . These data suggest that pretreatment or simultaneous coincubation of RU-ME with BPDE is required for inhibition of BPDE-induced activation of AP-1 and NF{kappa}B.



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  		Fig. 6. No inhibitory effect of RU-ME on activation of AP-1 (A) and NF{kappa}B (B) when added 3 h after cells were treated with BPDE. P+1–1 or NF{kappa}B mass1 cells were seeded into each well of 96-well plates and cultured as described in Fig. 1Citation . 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 NF{kappa}B activity in treated culture versus medium controls (23 24 25) . Each bar indicates the mean of four repeat wells; bars, ±SD.

 
Effect of the RU-ME Fraction on BPDE-induced DNA Adduct Formation.
Black raspberries contain multiple compounds with known chemopreventive activity (12) . Among these, ellagic acid has been shown to react with BPDE to form covalently linked cis and trans adducts in which the reactive epoxide ring of the pyrene is open, rendering the BPDE harmless (37) . To determine whether inhibition of BPDE-induced activation of AP-1 and NF{kappa}B by the RU-ME fraction might be because of a similar reaction of compounds in RU-ME with BPDE, we tested the effect of RU-ME on BPDE-induced DNA adduct formation. If compounds in RU-ME react with BPDE, then one might expect lowered levels of BPDE binding to Cl 41 cell DNA. To determine the effect of RU-ME on BPDE-DNA adduct formation, cultured Cl 41 cells were treated with [3H]BPDE or [3H]BPDE and RU-ME mixture. The 3H count in a known quantity of purified genomic DNA was determined. The number of BPDE-induced DNA adducts in a 10-kb genomic DNA fragment was then calculated. The results in Fig. 7Citation demonstrated that preincubation of the RU-ME fraction with BPDE did not reduce BPDE-DNA adduct formation in Cl 41 cells (Fig. 7)Citation . In fact, RU-ME treatment resulted in an increase in BPDE-DNA adduct formation.



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  		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).

 
Inhibition of AP-1 by RU-ME Is Attributable to Blocking BPDE-induced MAPK Activation.
AP-1 has been identified as a target of the MAPK family, including ERKs, JNKs, and p38 kinase (35) . To test the effects of the RU-ME fraction on BPDE-induced activation of the ERKs, JNKs, and p38 kinases in Cl 41 cells, the effects of RU-ME on phosphorylation of the MAPK family were tested. The results showed that pretreatment of cells with RU-ME led to a significant inhibition of phosphorylation of ERKs, JNKs, and p38 kinase (Fig. 8A)Citation , suggesting that all three of the MAPK family members are involved in the inhibitory effect of RU-ME on AP-1 activation.



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  		Fig. 8. Inhibition of BPDE-induced activation of MAPKs (A), and I{kappa}B{alpha} 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) I{kappa}B{alpha}. 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 NF{kappa}B by RU-ME Is Attributable to Its Influence on BPDE-induced Phosphorylation and Degradation of I{kappa}B{alpha}.
NF{kappa}B exists in cells in an inactive state by forming a complex with I{kappa}B{alpha}, an inhibitor of NF{kappa}B (27 , 28) . Signals leading to I{kappa}B{alpha} phosphorylation trigger the release of NF{kappa}B from I{kappa}B, resulting in an activation and translocation of NF{kappa}B from the cytoplasm to the nucleus, where it binds to promoter regions of its target genes (27 , 28) . To determine whether inhibition of BPDE-induced NF{kappa}B by RU-ME is caused by inhibition of I{kappa}B{alpha} phosphorylation and degradation, we determined I{kappa}B{alpha} phosphorylation in cells exposed to BPDE and RU-ME using phosphospecific antibody. Our results indicate that pretreatment of cells with RU-ME inhibited BPDE-induced increase in phosphorylation of I{kappa}B{alpha} at 90 min and degradation of I{kappa}B{alpha} protein at 270 min after BPDE treatment (Fig. 8B)Citation .


   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
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 NF{kappa}B 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 NF{kappa}B 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 NF{kappa}B 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 NF{kappa}B.

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) .

NF{kappa}B 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 I{kappa}B, an inhibitor for NF{kappa}B (27 , 28) . The signal leading to I{kappa}B phosphorylation triggers the release of NF{kappa}B from I{kappa}B, resulting in the activation and translocation of NF{kappa}B from the cytoplasm to the nucleus where NF{kappa}B binds to the promoter region of its specific targeting genes (27 , 28) . Similar to AP-1, NF{kappa}B 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 {alpha}, UV radiation, metals, reactive oxygen species, and HER-2/neu (31) . The two members of the NF{kappa}B family, v-rel and p52/lyt-10, and I{kappa}B family member Bcl-3 are potentially oncogenic (24) . H-Ras and Raf-1 can also activate NF{kappa}B in transfection assay (32 , 33) . Overexpression of the NF{kappa}B inhibitor, I{kappa}B{alpha}, 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 NF{kappa}B. 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 NF{kappa}B activities. The inhibitory effects of RU-ME on BPDE-induced activation of AP-1 and NF{kappa}B appears to be mediated via inhibition of MAPK activation and I{kappa}B{alpha} 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 NF{kappa}B, 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 p70S6k-independent pathways. Because BPDE is capable of inducing oxidative DNA damage, we speculate that BPDE-induced activation of AP-1 and NF{kappa}B 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 NF{kappa}B 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. Back

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 Back

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

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; I{kappa}B{alpha}, inhibitory subunit {kappa}-B; IL, interleukin; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase; NF{kappa}B, nuclear factor {kappa}B; P+, tumor promoter-sensitive; P-, tumor promoter-resistant; RU, Rubus occidentalis; TPA, 12-O-tetradecanoylphorbol-13-acetate; DPBS, Dulbecco’s PBS. Back

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

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

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

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


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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