Cytochrome P450 1B1, a novel chemopreventive target for benzo[a]pyrene-initiated human esophageal cancer

Cancer Letters 246 (2007) 109–114 www.elsevier.com/locate/canlet

Cytochrome P450 1B1, a novel chemopreventive target for benzo[a]pyrene-initiated human esophageal cancer Xia Wen, Thomas Walle * Department of Cell and Molecular Pharmacology and Experimental Therapeutics, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250505, Charleston, SC 29425, USA Received 22 December 2005; received in revised form 1 February 2006; accepted 2 February 2006

Abstract Esophageal cancer is common worldwide, with poor prognosis. Smoking, including exposure to polyaromatic hydrocarbons like benzo[a]pyrene (BaP), is a major risk factor. In human esophageal HET-1A cells, we found that time-dependent BaP–DNA binding was associated with upregulation of CYP1B1, but not CYP1A1, mRNA and protein. The dietary flavonoid 5,7dimethoxyflavone significantly inhibited BaP–DNA binding and down-regulated BaP-induced CYP1B1 mRNA and protein. 3 0 ,4 0 Dimethoxyflavone was an even more potent inhibitor of CYP1B1 expression, while resveratrol had no effect. Thus, dietary methoxylated flavones inhibited BaP-induced CYP1B1 transcription in a cell-specific manner and hold promise as chemopreventive agents in esophageal carcinogenesis. q 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Esophageal carcinogenesis; Benzo[a]pyrene; CYP1B1; Methoxylated flavones

1. Introduction Esophageal cancer, like oral cancer, occurs worldwide with its most common form being squamous cell carcinoma (SSC). In Europe and North America, the majority of esophageal and oral SCCs are caused by tobacco smoke and alcohol [1], with exposure to polyaromatic hydrocarbons (PAHs) like benzo[a]pyrene (BaP), as well as nitrosamines and other chemicals, considered the main risk factors [1,2]. Although the carcinogenic process is incompletely understood, tobacco is thought to cause cancers via the formation of carcinogen–DNA adducts. The formation of these adducts has been shown to be closely correlated with the mutation frequencies of the p53 tumor suppressor * Corresponding author. Tel.: C1 843 792 2507; fax: C1 843 792 2475. E-mail address: [email protected] (T. Walle).

gene [3,4]. The adduct formation is, however, critically dependent on the levels of the BaP bioactivating enzymes. In a previous study we showed that the tobacco carcinogen BaP induced CYP1B1 expression, and to a much lesser extent CYP1A1 expression, the enzymes responsible for the BaP–DNA binding, in human oral epithelial SCC-9 cells [5]. In the present study, we examined the effect of BaP on BaP–DNA binding as well as on CYP1B1 and CYP1A1 expression in HET-1A cells, a normal but immortalized human esophageal epithelial cell line [6]. A diet rich in fruits and vegetables has been shown to have a preventive effect on esophageal cancers [7,8]. As the two plant-based methoxylated flavones 5,7dimethoxyflavone (5,7-DMF) and 3 0 ,4 0 -dimethoxyflavone (3 0 ,4 0 -DMF) have been shown to decrease BaP– DNA binding by inhibition of CYP1B1/1A1 activity and/or protein expression in other human cells [9,10], we examined their effects, compared to resveratrol, on

0304-3835/$ - see front matter q 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.02.003

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BaP–DNA binding and the related molecular events in these esophageal cells. 2. Materials and methods 2.1. Chemicals 5,7-DMF and 3 0 ,4 0 -DMF were purchased from Indofine Chemical Co. (Somerville, NJ). Ethoxyresorufin, resorufin, resveratrol and BaP were obtained from Sigma Chemical Co. (St. Louis, MO). The purity of these chemicals was 97–99.5%. Williams’ Medium E was obtained from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum was obtained from Atlas Biologicals (Fort Collins, CO) and other cell culture components were purchased from Cellgro, Mediatech (Herndon, VA). [G-3H]BaP (76 Ci/mmol, radiochemical purity 99%) was purchased from Amersham Biosciences (Piscataway, NJ). 2.2. Cell culture and treatment Normal human esophageal epithelial HET-1A cells [6] were a gift from Dr Gary Stoner at Ohio State University. The cells were cultured at 37 8C in an atmosphere of 5% carbon dioxide and 90% relative humidity in Dulbecco’s Minimum Essential Medium with high glucose (4.5 g/l) and 2% fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 5 mg/ml insulin, 5 mg/ml transferrin, 0.01 mg/ml hydrocortisone, and 0.01 mg/ml cholera toxin, as previously described [11]. Two to three days after seeding in 6-well plates, 90% confluent cells were treated with 1 mM BaP in the presence or absence of 5,7-DMF, 3 0 ,4 0 -DMF or resveratrol in growth medium for the times and concentrations indicated in the figures. The concentrations of 5,7-DMF, 3 0 ,4 0 -DMF and resveratrol used, 20 mM, had no cytotoxic effects (data not shown). Although dietary polyphenol concentrations vary widely, 20 mM can be considered an achievable concentration in the aerodigestive lumen [12]. Dimethyl sulfoxide (DMSO, 0.1% of final volume) was used as a vehicle control in all experiments. 2.3. BaP–DNA binding The binding of BaP to cellular DNA was measured as previously described [13]. HET-1A cells cultured in six-well plates were treated with 1 mM [3H]BaP (10 mCi/ml medium) for 30 min (control) to 72 h. In the inhibitory experiments, the cells were co-treated with 1 mM [3H]BaP and 20 mM 5,7DMF for 24 h. After treatment, the cells were washed, detached with lift buffer and pelleted. Cell pellets were then lysed in polyamine swell buffer and spun down to get crude nuclear pellets [14]. The nuclei were purified by centrifugation through a 30% sucrose cushion. Nuclear protein and RNA were digested with proteinase K and RNase, and the samples were extracted repeatedly with phenol/chloroform.

DNA was then precipitated and washed with cold ethanol. The amount and purity of DNA in the dissolved pellet were measured by UV and the amount of [3H]BaP bound to DNA was quantified by liquid scintillation spectrometry. 2.4. Western blot analysis of CYP1A1 and 1B1 HET-1A cells grown in 100 mm dishes were treated with 1 mM BaP in the presence or absence of 20 mM 5,7-DMF, 3 0 ,4 0 -DMF or resveratrol for 24 h. After treatments, the cells were washed and scraped into tubes, resuspended in buffer with protease inhibitors and sonicated, as previously described [5,9]. The microsomal fraction was isolated by differential centrifugation at 4 8C at 10,000 and 100,000!g. Denatured microsomal proteins (30 mg) were separated on 10% NuPAGE Novex Bis–Tris gels (Invitrogen, Carlsbad, CA), transferred to nitrocellulose, blocked and incubated with primary antibodies to CYP1A1 (rabbit anti-trout 1A from Biosense Laboratories, Bergen, Norway) or CYP1B1 (rabbit anti-human 1B1, BD Gentest, Woburn, MA), as described [5,9]. After washing, incubation with goat anti-rabbit IgG peroxidase conjugate and incubation with chemiluminescent substrate, the membranes were exposed to ECL film. Baculovirus-expressed human CYP1A1 (0.12 mg) and CYP1B1 (0.32 mg) supersomes (BD Gentest, Bedford, MA) were used as standards. All blots were done at least three times. 2.5. CYP1A1 and 1B1 mRNA analysis HET-1A cells cultured in 96-well plates were treated with BaP and/or 5,7-DMF, 3 0 ,4 0 -DMF or resveratrol as mentioned above for Western blot analysis. The quantitative detection of CYP1A1 and 1B1 mRNA in cell lysates was determined using branched DNA (bDNA) technology [15] with primers for human CYP1A1, CYP1B1 and GAPDH, as previously described [5] (QuantiGene kits, Genospectra Co, Fremont, CA). Briefly, target mRNA captured in coated microwells was amplified with branched oligonucleotide probes with attached alkaline phosphatase. After the addition of chemilumigenic substrate, the luminescence was quantified with a plate reader. Six wells were used for each treatment with each sample normalized to its GAPDH mRNA content. 2.6. Data analysis Statistical differences between different treatments were determined using two-tailed unpaired ANOVA with a multiple comparison (Dunnett) post-test (InStat). The results were expressed as meansGSD for at least triplicate determinations.

3. Results The formation of carcinogen–DNA adducts plays an important role for the initiation of human cancers such

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cells, but was clearly visible after BaP treatment (Fig. 1C). In the presence of 5,7-DMF, the BaP–DNA binding was markedly inhibited (Fig. 2A). The same concentration of 5,7-DMF also inhibited the BaP-induced part of the CYP1B1 mRNA expression but did not affect the baseline levels (Fig. 2B). Also, the BaP-induced CYP1B1 protein was blocked by 5,7-DMF treatment (Fig. 2C). When extending the study to include one additional methoxylated polyphenol, i.e. 3 0 ,4 0 -DMF, it demonstrated a more potent inhibition of BaP-induced CYP1B1 mRNA to well below baseline values

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Fig. 1. Effects of BaP on BaP–DNA binding (A), CYP1B1/1A1 mRNA levels (B) as well as their protein expression (C) in the HET1A cells. The cells were exposed to 1 mM 3[H]BaP for 0.5–72 h (A) or 1 mM BaP or vehicle (DMSO) for 24 h (B and C). Values in A and B are meanGSD (nZ6). The Western blot shown in (C) is a representative example of three independent experiments with bactin as a loading control. The filled bars in (B) represent CYP1B1 mRNA and the open bars CYP1A1 mRNA. *Significantly higher than 0.5 h BaP treatment (A) or DMSO treatment (B), P!0.05.

B CYP1B1 mRNA level (CYP1B1/GAPDH ratio)

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as esophageal cancer, therefore, BaP–DNA binding levels were determined in the HET-1A cells. After a low concentration of BaP (1 mM) treatment (Fig. 1A), BaP–DNA binding was increased in a time-dependent manner, with a 40-fold increase after 72 h treatment compared to 0.5 h. The increase of BaP–DNA binding over time indicated that BaP-bioactivating enzymes were expressed in these cells. CYP1A1 mRNA was not expressed in the HET-1A cells and was not inducible by BaP. This was also true for the CYP1A1 protein. On the other hand, CYP1B1 mRNA was constitutively expressed in these cells and was induced about 2-fold by BaP (Fig. 1B). Interestingly, CYP1B1 protein could not be detected in the untreated

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β–actin Fig. 2. Effect of 5,7-DMF on BaP-induced DNA binding (A), CYP1B1 mRNA levels (B) as well as its protein expression (C) in the HET-1A cells. The cells were treated for 24 h with 1 mM BaP or vehicle (DMSO) in the presence or absence of 20 mM 5,7-DMF. Values are meanGSD (nZ3). The Western blot shown in (C) is a representative example of three independent experiments with b-actin as a loading control. *Significantly different from 24 h BaP treatment, P!0.05.

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A CYP1B1 mRNA level (CYP1B1/GAPDH ratio)

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β−actin Fig. 3. Effects of 3 0 ,4 0 -DMF and resveratrol on CYP1B1 mRNA levels (A) as well as its protein expression (B) in HET-1A cells. The cells were treated for 24 h with DMSO, 1 mM BaP alone or 1 mM BaP and 20 mM 3 0 ,4 0 -DMF or resveratrol (Res). Values are meanGSD (nZ6). The Western blot shown in (B) is a representative example of 3 independent experiments with b-actin as a loading control. *Significantly different from 24 h BaP treatment. P!0.05.

(Fig. 3B). Surprisingly, the well-accepted chemopreventive polyphenol resveratrol had no effect on BaP-induced CYP1B1 expression mRNA expression or protein expression in the HET-1A cells. A previous study had determined the 5,7-DMF uptake in the oral SCC-9 cells, reaching a steady-state level of about 2.6 nmol/mg protein after 10 min, i.e. about a 20-fold accumulation [5]. Repeating this experiment in the HET-1A cells demonstrated a similar time-course of uptake, but with a greater accumulation, up to about 8 nmol/mg protein (data not shown). 4. Discussion In the present study, a low concentration of BaP dramatically induced BaP–DNA binding in a timedependent manner in the immortalized normal esophageal HET-1A cells with a 40-fold increase at 72 h compared to 0.5 h (Fig. 1A). This was very similar to our previous findings in oral epithelial carcinoma SCC9 cells [5], suggesting a comparable complement of BaP bioactivating enzymes in the two cell types as well as a similar initiating effect of BaP in esophageal and oral cancers. In the oral cells, BaP caused preferential induction of CYP1B1 with some induction of CYP1A1, as supported by both mRNA and protein measurements. In the HET-1A cells, CYP1B1 mRNA

was constitutively expressed and BaP caused an about 2-fold induction (Fig. 1B). This effect resulted in clear expression of the CYP1B1 protein (Fig. 1C). In contrast, the constitutive CYP1A1 mRNA levels were extremely low and unaffected by BaP (Fig. 1B). CYP1A1 protein was undetectable with or without BaP treatment (Fig. 1C). Moreover, the EROD assay, a highly sensitive method preferentially for CYP1A1 catalytic activity [16], showed very low baseline values and was not induced by BaP treatment. These observations suggest CYP1B1 as a specific and early biomarker as well as a promising chemopreventive target in PAH-induced esophageal cancer. Interestingly, the similar BaP–DNA binding level in HET-1A and oral SCC-9 cells cannot be completely explained by induction of CYP1B1, as the induced CYP1B1 mRNA levels in the HET-1A cells were much lower than in the SCC-9 cells. The low expression of CYP1B1 protein in both cell lines may be similar, although a direct quantitative comparison could not be made. However, the involvement of additional BaP bioactivating factors in the esophageal cells can not be excluded [17]. The expression of CYP1 enzymes in human esophageal tissue specimens has been reported previously at a time when optimal analytical technology may not yet have been developed for the individual isoforms. In one

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study, CYP1A protein expression was present in a small fraction of non-neoplastic samples (3 of 10 cases), but it was more frequently observed in esophageal tumor specimens (32 of 50 cases) [18]. Using a different antibody, Nakajima et al. found that CYP1A1/1A2 protein was expressed at significantly higher levels in all esophageal tumors vs. normal tissues [19]. In two other studies, CYP1A1 mRNA was detected in the majority (84%) of non-tumorous specimens from esophageal cancer patients [20] and in all Barrett’s esophagus and some matching normal tissue [21]. However, none of these studies addressed CYP1B1 expression, which should be much more important in BaP-induced esophageal cells, as indicated by the present study. The minimal to undetectable CYP1A1 mRNA and protein and catalytic activity in untreated HET-1A cells in the present study may be due to the noncancerous nature of these esophageal epithelial cells. Also, based on previous comparisons [16,22], the contribution by CYP1A2 to BaP bioactivation is much less than by CYP1A1 and 1B1 and, thus, its possible role in BaP-induced DNA binding was not investigated. Regarding PAH-induced CYP1B1 as a chemopreventive target in the esophageal epithelium, the methoxylated flavone 5,7-DMF effectively inhibited BaP–DNA binding (Fig. 2A). The mechanism of this effect may be both downregulation of CYP1B1 protein expression and direct inhibition of its enzyme activity. First, the BaP-induced CYP1B1 mRNA (Fig. 2B) and protein expression (Fig. 2C) were both completely inhibited. Previously, 5,7-DMF was shown to inhibit the transcription of CYP1A1 in both hepatic [9] and oral [5] cells. However, 5,7-DMF was unable to inhibit the transcription of CYP1B1 in the oral cells. This dramatic difference in responses between oral and esophageal cells has no clear explanation. It might be related to cell specificity in some aspect of the transcriptional regulation of CYP1B1, as has been addressed in a previous study [5] and more generally by Zhang et al. [23]. Second, we have previously shown that 5,7-DMF is a very potent direct inhibitor of the catalytic activity of the CYP1B1 protein [5]. Thus, a Ki value as low as 0.58 mM was found for this mixed-type inhibition. Considering the extensive epithelial cell accumulation of 5,7-DMF in the HET-1A cells, these intracellular concentrations are easily achievable. When expanding our study to two other polyphenols, 3 0 ,4 0 -DMF, even more dramatically than 5,7-DMF, inhibited the BaP-induced CYP1B1 mRNA expression in the HET-1A cells. In sharp contrast, resveratrol, an effective inhibitor of BaP-induced CYP1B1 mRNA in human bronchial epithelial cells [24], was without

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effect in the HET-1A cells. This, again, points to cell selective effects of the polyphenol CYP1B1 inhibitors. The mechanism by which 5,7-DMF and 3 0 ,4 0 -DMF affect CYP1B1 transcription in this study is not clearly understood. First, it has been shown that BaP induces CYP1B1 expression by transactivating the aryl hydrocarbon receptor (AhR) [25]. Of the two methoxylated flavones, 3 0 ,4 0 -DMF has been shown to be an AhR antagonist [26]. However, 3 0 ,4 0 -DMF as well as 5,7-DMF may exert their inhibitory effects on CYP1B1 transcription by affecting a number of other less well understood transcriptional factors such as hsp90 [27] or Sp1 [28]. Another regulation mechanism may involve promoter/ enhancer hypomethylation of CYP1B1 the gene [29]. The bioavailability of many abundant dietary polyphenols, e.g. quercetin [30–32], chrysin [33] and resveratrol [34], in humans is very low. This is mainly due to rapid and extensive glucuronidation and sulfation in the intestine/liver. In contrast, the methylated flavones studied here, i.e. 5,7-DMF and 3 0 ,4 0 DMF, must first be oxidatively demethylated and may, therefore, have much improved bioavailability. This is supported by a recent metabolic study [35]. Both 5,7DMF and 3 0 ,4 0 -DMF are natural products [36,37] with at least 5,7-DMF being present in plants used for human consumption. Thus, effective inhibition of carcinogenactivating CYP1B1 combined with good bioavailability give these compounds the potential to be effective chemopreventive agents. This may be particularly true in oral and esophageal tissue, both demonstrating a very high accumulation of 5,7-DMF. Acknowledgements This study was supported by the Department of Defense/Hollings Cancer Center grant N6311602MD200 and the National Institutes of Health grant GM55561. It was also partially supported by a grant from the American Institute for Cancer Research (02A095). U. Kristina Walle is highly acknowledged for her efforts and skills in preparation of this manuscript. The HET-1A cells were a kind gift from Dr Gary D. Stoner at Ohio State University. References [1] G.P. Pfeifer, M.F. Denissenko, M. Olivier, N. Tretyakova, S.S. Hecht, P. Hainaut, Tobacco smoke carcinogens, DNA damage and p53 multations in smoking-associated cancers, Oncogene 21 (2002) 7435. [2] G.D. Stoner, A. Gupta, Etiology and chemoprevention of esophageal squamous cell carcinoma, Carcinogenesis 22 (2001) 1737.

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