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Developing a high-throughput system for the screening of cytochrome P450 1A1 – Inhibitory polyphenols
Toxicology in Vitro 21 (2007) 996–1002 www.elsevier.com/locate/toxinvit
Review
Developing a high-throughput system for the screening of cytochrome P450 1A1 – Inhibitory polyphenols Hau Yi Leung a
a,1
, Yun Wang
b,1
, Ho Yee Chan b, Lai K. Leung
a,b,*
Food and Nutritional Sciences Programme, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong b Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Received 6 January 2007; accepted 17 April 2007 Available online 27 April 2007
Abstract Polycyclic aromatic hydrocarbons (PAH) are established procarcinogens that can be found in our environment. The carcinogenicity of these compounds is initiated by their metabolic intermediates, and the extent of biotransformation determines the amount of reactive intermediates generated. CYP1A1 is a major enzyme that metabolizes PAH into carcinogenic moieties. Since previous studies have shown that increased CYP1A1 activity is associated with a higher cancer risk. Identifying CYP1A1-inhibitory compounds in diet or natural products are of genuine interest for chemoprevention studies. In this project, a stable cell line expressing human CYP1A1 was established for the screening of potential chemopreventive agents. Because of a lacking cellular transport mechanism in assays performed on recombinant protein, an ‘in-cell’ assay system might be a better estimate at the tissue level. Theaflavins were strong inhibitors of ethoxyresorufin-O-deethylase (EROD) activity when assayed on recombinant human CYP1A1 protein. However, this inhibition was not observed in the CYP1A1-expressing cells. The ‘in-cell’ IC50 values determined for compounds such as genistein, quercetin, chalcone, etc. were comparable to the values determined in recombinant protein. This ‘in-cell’ assay has the additional advantages of short sample processing time, and the tedious procedures of protein expression and purification can be waived. 2007 Elsevier Ltd. All rights reserved. Keywords: CYP1A1; Stable transfection; EROD
Contents 1. 2.
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemicals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cell culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Construction of CYP1A1 expression plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Establishing a stable cell line expressing CYP1A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: FBS, fetal bovine serum; AHR, aryl hydrocarbon receptor; XRE, xenobiotic response element; DMBA, 7,12-dimethylbenz[a]anthracene; PAH, polycylic aromatic hydrocarbon; B[a]P, benzo[a]pyrene; EROD, ethoxyresorufin-O-deethylase; DMSO, dimethyl sulfoxide; EGCG, epigallocatechin gallate. * Corresponding author. Present address: Department of Biochemistry, The Chinese University of Hong Kong, Rm. 507C MMW Bldg, Shatin, N.T., Hong Kong. Tel.: +852 26098137; fax: +852 26037732. E-mail address: [email protected] (L.K. Leung). 1 These authors contributed equally to this work. 0887-2333/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.04.005
H.Y. Leung et al. / Toxicology in Vitro 21 (2007) 996–1002
4.
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3.1. Enzyme inhibition assays performing in intact MCF-7 cells expressing CYP1A1 . . . . . . . . . . . . 3.2. Enzyme inhibition assays performing in recombinant CYP1A1 . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Enzyme kinetic studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Statistical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. EROD activity inhibited by polyphenols in cells over-expressing CYP1A1 . . . . . . . . . . . . . . . . . 4.2. Enzyme kinetic study of chalcone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Effects of selected tea polyphenols on EROD activity in recombinant CYP1A1 protein and cells Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
2. Materials and methods
Polycylic aromatic hydrocarbons (PAH) are common toxicants in our environment. They can be extracted from diesel exhaust, char broiled meat, tobacco smoke, overheated cooking oil, etc. (Environmental Protection Agency, 1990; International Agency for Research on Cancer, 1983). PAHs are classified as procarcinogens because these compounds are ultimately metabolized into DNA-attacking electrophiles in our body. Aryl hydrocarbon receptor (AHR) reconciles the transformation of these procarcinogens into genotoxic moieties in a series of reactions. Upon PAH activation, the AHR complex translocates from cytosol to the nucleus and interacts with an AHR nuclear translocator (ARNT). This protein complex docks onto a gene containing xenobiotic responsive elements (XRE) and initiates its transcription (Kronenberg et al., 2000). Cytochrome P450 1A1 (CYP1A1) is one of these downstream genes of AHR transactivation (Dertinger et al., 2000; Safe, 2001). The subsequent translated CYP1A1 enzyme biotransforms PAHs into DNA-attacking species. The observation that benzo[a]pyrene failed to induce CYP1A1 and liver cancer in AHR-knockout mice has illustrated this inter-relationship (Shimizu et al., 2000). Studies have shown that polymorphisms with higher activity of CYP1A1 are associated with increased risk of various cancers (Taioli, 1999). In contrast, the inhibition of CYP1 enzymes appears to be beneficial in the prevention of 7,12-dimethylbenz[a]anthracene (DMBA)–DNA adduct formation in vivo and in vitro (Kleiner et al., 2002; MacDonald et al., 2001). Developing a fast screening methodology for CYP1A1-inhibitory compounds is prompted by these findings. Previously we employed an XRE-driven reporter assay technique to assess CYP1 expression and found that the luciferase activity suppressed by polyphenols correlated to the reduced DMBA–DNA adduct formation (Chan and Leung, 2003). To complement the reporter assay, the present study aimed to establish a stable cell line for the screening of CYP1A1 inhibitor at the enzyme level.
2.1. Chemicals
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7,12-Dimethylbenz[a]anthracene (DMBA) and DMSO were obtained from Sigma–Aldrich Fine Chemicals (St Louis, MO, USA). Theaflavins were isolated as previously described (Leung et al., 2001). All other chemicals, if not stated, were acquired from Sigma Chem. 2.2. Cell culture MCF-7 cells (obtained from American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI – 1640 phenol red free media (Sigma–Aldrich) and 10% fetal bovine serum (Invitrogen Life Technology, Rockville, MD, USA) at 37 C and 5% carbon dioxide. 2.3. Construction of CYP1A1 expression plasmid Total RNA was isolated from cells grown in six-well plates (Costar). RNA of 1 lg was used for the first strand cDNA synthesis, and the final volume was diluted to 20 ll. Primers encompassed the full length of CYP1A1 were designed and a Perkin Elmer Thermocycler (GeneAmp PCR System 2400) was utilized to amplify the target cDNA. A PCR reaction consisted of 0.2 mmol/l dNTP, 2 ll cDNA, 0.2 lmol/l of each primer, 1 · PCR buffer and 1 U of pfu polymerase (Invitrogen). The conditions were 94 C for 45 s, 65 C for 45 s, 72 C for 1 min and a final extension period of 7 min at 72 C for 25 cycles. The PCR product was separated on a 1.8% agarose gel, stained with ethidium bromide, and extracted into a 1.5 ml eppendorf tube for enzyme digestion. The digested fragment was inserted into the mammalian expression vector pcDNA3.1 (Promega) and the cDNA sequence of hCYP1A1 was confirmed subsequently. 3. Establishing a stable cell line expressing CYP1A1 The expression construct was transfected into MCF-7 cells by lipofectamine (Invitrogen) and selected by geneticin
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H.Y. Leung et al. / Toxicology in Vitro 21 (2007) 996–1002
activities were expressed as a relative EROD activity to the control. 3.2. Enzyme inhibition assays performing in recombinant CYP1A1 Recombinant CYP1A1 proteins expressed in microsomes (Supersomes) were purchased from BD Gentest Corp. (Woburn, MA, USA). Chalcone and tea polyphenols were tested using recombinant CYP1A1 for comparison. Protein of 2 pmol was incubated in 100 ll PBS, pH 7.2 with 400 nM ethoxyresorufin and polyphenol in different concentrations. The reaction was initiated by 500 lM NADPH, and terminated by 100 ll of ice-cold methanol after 20 min of incubation. The fluorescence was measured as described earlier. Fig. 1. Establishing a stable cell line expressing CYP1A1 MCF-7 cells were transfected with CYP1A1 expression vector and selected. RT-PCR was used to analyze the expression of clones A1–A12 as shown in Fig. 1a. EROD activity of the clones was also performed to verify the mRNA expression (Fig. 1b).
(Invitrogen). After maintaining the transfected cells for 21 days, clones were examined for the message expression and activity by RT-PCR (Fig. 1a) and ethoxyresorufin-Odeethylase (EROD) activity (Fig. 1b). Clones that consistently had the highest expression and activity were selected for the establishment of a stable CYP1A1 expressing cell line. Clones A5 and A10 displayed a distinct expression of CYP1A1 in RT-PCR, and subsequent EROD assay confirmed that Clone A10 had an enzyme activity 14 times of those clones transfected with empty vector. Contrasting to a 2-fold increase in cells treated with DMBA (Chan et al., 2003b), these Clone A10 cells designated as MCF-7CYP1A1 provided an excellent alternative to xenobiotic-induced model for the screening of CYP1A1-inhibitory compounds. 3.1. Enzyme inhibition assays performing in intact MCF-7 cells expressing CYP1A1 The assay method was performed as previously described (Chan et al., 2003a) with some modifications. In brief, MCF-7CYP1A1 cells were seeded in 96-well plates for 24 h. The medium was then removed and EROD activity assays were then carried out. Ethoxyresorufin (5 lM) and salicylamide (1.5 mM) of 50 ll were dissolved in PBS, and tested phytochemicals were added in wells and incubated at 37 C for 15 min. The reactions were stopped by 50 ll of ice-cold methanol, and the resorufin generated was measured by a FLUOstar Galaxy microplate reader (BMG Labtechnologies, Offenburg, Germany) with 544 nm and 590 nm as the respective excitation and emission wavelengths. The activities were quantified against resorufin standards. In order to avoid a batch-to-batch variation in cell number and viability, timing, etc., the
3.3. Enzyme kinetic studies All the above conditions, described in assays performing in cell or recombinant protein, were applied except that many substrate concentrations were tested. 3.4. Statistical methods A SigmaPlot software package (IL, USA) was utilized for statistical analysis. The results, whenever applicable, were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test if significant differences (p < 0.05) were observed. T-test was performed for the other comparisons. For the IC50 estimations, the non-linear curve-fitting program installed in the software was employed. 4. Results 4.1. EROD activity inhibited by polyphenols in cells overexpressing CYP1A1 Some flavonoids were tested on the CYP1A1 inhibition in the MCF-7CYP1A1 cells (Fig. 2). The inhibitory results of chalcones, isoflavones, flavonols and other phytochemicals on EROD activity are shown in Fig. 2a–c, and the estimated IC50 are listed in Table 1. Among all types of flavonoids tested, the flavonol quercetin and kaempferol displayed the strongest inhibitory effect on CYP1A1. In the isoflavone category, baicalein was the most potent, followed by genistein, equol and biochanin A. Daidzein did not inhibit the EROD activity in the current study. 2 0 OH and 2-OH chalcones had the lowest IC50 values, whereas butein was the weakest. 4.2. Enzyme kinetic study of chalcone Enzyme kinetic assays on the polyphenol chalcone were carried out in MCF-7CYP1A1 cells and recombinant protein for comparison. The Ki’s (Fig. 3b and e), which were
H.Y. Leung et al. / Toxicology in Vitro 21 (2007) 996–1002
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Fig. 2. Inhibitory action of polyphenols on CYP1A1 in MCF-7CYP1A1 cells. The inhibitory effects of chalcones, isoflavones and flavonols and other phytochemicals were determined in CYP1A1 stable cells as shown in Fig. 2a, 2b and 2c, respectively. Values are means ± S.D., n = 3. Table 1 Estimated IC50 values from MCF-7CYP1A1 ‘in-cell’ assays Flavonoids Chalcones: 2 0 -OH chalcone 2-OH chalcone 4-OH chalcone 4,2 0 ,4 0 -trihydroxychalcone Butein Isoflavones: Equol Daidzein Genistein Biochanin A Baicalein
Estimated IC50 (lM) 11.14 8.64 16.14 13.85 25.00 >44.44 50.00 33.56 24.44 10.43
Flavonols: Quercetin Kaempferol
1.36 5.43
Others: Luteolin Resveratrol Naringenin
6.74 36.11 >100.00
recombinant protein assay. The ‘in-cell’ assaying method could be applied for enzyme kinetic analysis.
Estimated ki (lM) 0.38 0.12 4.10 5.20 8.50 – – 15.35 4.00 0.40 0.20 0.30
4.3. Effects of selected tea polyphenols on EROD activity in recombinant CYP1A1 protein and cells EROD activity was determined in intact MCF-7CYP1A1 cells and in recombinant protein (Supersomes BD Gentest, Wobash, MA). Some tea polyphenols displayed strong inhibition on EROD activity assayed in Supersomes. The IC50 values were estimated to be 65 lM for theaflavin 1 (TF1), theaflavin 3 0 -gallate (TF2) and theaflavin-3,3 0 -digallate (TF3), and about 30 lM for epigallocatechin gallate (EGCG) (Fig. 4a). However, the EROD activity was neither significantly suppressed in DMBA-induced cells (Fig. 4b) nor in MCF-7CYP1A1 cells (Fig. 4c). 5. Discussion
0.50 11.40 –
derived from the Lineweaver–Burk plot (Fig. 3a and c), were determined to be 0.5 lM in-cell assay and 1.4 lM in
With regard to the CYP1A1-inhibitory activities performed in stable cells, the trend of data was consistent with that performed in recombinant protein as we previously reported (Chan and Leung, 2003; Wang et al., 2005). The estimated IC50 values of reverstrol, quercetin, kaempferol and naringenin in the present study are greater than the
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Fig. 3. ‘In-cell’ enzyme kinetic determination EROD assays were performed on MCF-7CYP1A1 cells treated with 0, 0.5, 1, 5 and 10 lM of chalcone. The Lineweaver–Burk plot (Fig. 3a) was generated from the reciprocal data, and the value of Ki was derived from the slopes of the regression lines (Fig. 3b). For comparison, Fig. 3c and 3d are Lineweaver–Burk plot and Ki derivation from data obtained from recombinant protein. Values shown are mean ± S.D., n = 3.
values obtained from recombinant protein assays (Schwarz and Roots, 2003) by 2- to 20-fold. Although the ‘in-cell’ assay tends to over-estimate the IC50 values, the method can still be a viable substitution for recombinant protein. The differences, which could be attributed to the cellular transport or metabolism of the administered chemicals, would be a better predictor at the physiological level. Among the green tea catechins, (–)-epigallocatechin gallate (EGCG) has stronger and higher anti-proliferative, anti-neoplastic and anti-mutagenic activities than (–)-epicatechin (EC) and (–)-epicatechin gallate (EGC) (Ahmad and Mukhtar, 1999; Liang et al., 1997; Mukhtar and Ahmad, 1999). Oral or topical administration of EGCG inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK)-induced lung tumors (Xu et al., 1992), azoxymethane (AOM)-induced colon cancer (Ohishi et al., 2002), and DMBA-induced breast cancer (Kavanagh et al., 2001). These studies indicate that EGCG is protective against chemical-induced carcinogenesis. Other studies have demonstrated that EGCG can inhibit the biotransformation of carcinogens by modulating CYP1A enzyme expression and activities, and decreasing the production of ultimate carcinogens (Williams et al., 2000; Xu et al., 1992). However, our results performed on MCF-
7CYP1A1 cells suggested that EGCG was merely a weak inhibitor on CYP1A1. Theaflavins also exhibit anti-carcinogenesis effects in different models, and little is known about the mechanism of its chemoprevention. They inhibit neoplastic transformation in human lung cells (Sazuka et al., 1997), mammary cells and rat tracheal epithelial cells (Steele et al., 2000). Similar to green tea polyphenols, they are strong antioxidants (Leung et al., 2001) and are capable of inhibiting DNA-carcinogen binding (Schut and Yao, 2000). Others have suggested that theaflavins may play a role in modulating carcinogens activation (Apostolides et al., 1997). Black tea has been demonstrated to be inhibitory in benzo[a]pyrene (B[a]P) metabolism in a cell-free system (Hammons et al., 1999) and in a hepatocarcinoma cell culture model (Feng et al., 2002). Our screening results also indicated that theaflavins displayed minimal CYP1A1-inhibition at the cellular level. Since theaflavins have high molecular weight, they might not be able to reach the vicinity in interacting with the enzyme. In a recent case–control study quercetin consumption has been associated with reduced lung cancer risk in Hawaii (Marchand et al., 2000). Compared to an IC50 value of 0.2 lM determined in recombinant protein
H.Y. Leung et al. / Toxicology in Vitro 21 (2007) 996–1002
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Fig. 4. Differential inhibitory effects of tea polyphenols on EROD activity in recombinant CYP1A1 protein and cells CYP1A1 inhibitory effects of tea polyphenols – epigallocatechin gallate (EGCG), theaflavin 1 (TF1), theaflavin-3,3 0 -digallate (TF2) and theaflavin 3 0 -gallate (TF3) – were assayed in different systems. Fig. 4a represents the CYP1A1-inhibitory results of some tea polyphenols performed in recombinant protein. Fig. 4b illustrates the inhibition assay carried out in DMBA-induced MCF-7 cells. Fig. 4c indicates the ‘in-cell’ assay performed in MCF-7CYP1A1 cells. Values are means ± SD, n = 3.
(Schwarz et al., 2005), the value of quercetin was determined to be 1.36 lM in the present study. Quercetin was the most effective suppressor of CYP1A1 among various dietary chemicals tested in the present study. In addition quercetin is an agonist of AHR (Ciolino et al., 1999), it may compete for the binding of AHR and reduce the expressions of CYP1A1. Quercetin appears to be a potent dietary phytochemical in the protection against PAH toxicity. In summary, this study indicated that CYP1A1 stable cell line could be a replacement for the recombinant protein system. Because the assays can be performed in 96-well plates and no protein purification is required, this stable cell line is a better alternative for the screening of dietary CYP1A1-inhibitory compounds. References Ahmad, N., Mukhtar, H., 1999. Green tea polyphenols and cancer: Biologic mechanisms and practical implications. Nutr. Rev. 57 (3), 78– 83. Apostolides, Z., Balentine, D.A., Harbowy, M.E., Hara, Y., Weisburger, J.H., 1997. Inhibition of PhIP mutagenicity by catechins, and by theaflavins and gallate esters. Mutat. Res. 389 (2–3), 167–172. Chan, H.Y., Leung, L.K., 2003. A potential protective mechanism of soya isoflavones against 7,12-dimethylbenz[a]anthracene tumour initiation. Br. J. Nutr. 90 (2), 457–465.
Chan, H.Y., Wang, H., Leung, L.K., 2003a. The red clover (Trifolium pratense) isoflavone biochanin A modulates the biotransformation pathways of 7,12-dimethylbenz[a]anthracene. Br. J. Nutr. 90 (1), 87– 92. Chan, H.Y., Wang, H., Tsang, D.S., Chen, Z.Y., Leung, L.K., 2003b. Screening of chemopreventive tea polyphenols against PAH genotoxicity in breast cancer cells by a XRE-luciferase reporter construct. Nutr. Cancer 46 (1), 93–100. Ciolino, H.P., Daschner, P.J., Yeh, G.C., 1999. Dietary flavonols quercetin and kaempferol are ligands of the aryl hydrocarbon receptor that affect CYP1A1 transcription differentially. Biochem. J. 340 (Pt 3), 715–722. Dertinger, S.D., Lantum, H.B., Silverstone, A.E., Gasiewicz, T.A., 2000. Effect of 3 0 -methoxy-4 0 -nitroflavone on benzo[a]pyrene toxicity. Aryl hydrocarbon receptor-dependent and -independent mechanisms. Biochem. Pharmacol. 60 (2), 189–196. Environmental Protection Agency. 1990. Aerometric Information Retrieval System (AIRS). Data for 1985–1990. Feng, Q., Torii, Y., Uchida, K., Nakamura, Y., Hara, Y., Osawa, T., 2002. Black tea polyphenols, theaflavins, prevent cellular DNA damage by inhibiting oxidative stress and suppressing cytochrome P450 1A1 in cell cultures. J. Agric. Food Chem. 50 (1), 213–220. Hammons, G.J., Fletcher, J.V., Stepps, K.R., et al., 1999. Effects of chemoprotective agents on the metabolic activation of the carcinogenic arylamines PhIP and 4-aminobiphenyl in human and rat liver microsomes. Nutr. Cancer 33 (1), 46–52. International Agency for Research on Cancer. 1983. Polynuclear aromatic compounds, Part 1, Chemical, environmental and experimental data. IARC Monogr. Eval. Carcinog. Risk Chem. Hum. 32, 1–453.
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Kavanagh, K.T., Hafer, L.J., Kim, D.W., et al., 2001. Green tea extracts decrease carcinogen-induced mammary tumor burden in rats and rate of breast cancer cell proliferation in culture. J. Cell Biochem. 82 (3), 387–398. Kleiner, H.E., Vulimiri, S.V., Reed, M.J., Uberecken, A., DiGiovanni, J., 2002. Role of cytochrome P450 1A1 and 1B1 in the metabolic activation of 7,12-dimethylbenz[a]anthracene and the effects of naturally occurring furanocoumarins on skin tumor initiation. Chem. Res. Toxicol. 15 (2), 226–235. Kronenberg, S., Esser, C., Carlberg, C., 2000. An aryl hydrocarbon receptor conformation acts as the functional core of nuclear dioxin signaling. Nucleic Acids Res. 28 (12), 2286–2291. Leung, L.K., Su, Y., Chen, R., Zhang, Z., Huang, Y., Chen, Z.Y., 2001. Theaflavins in black tea and catechins in green tea are equally effective antioxidants. J. Nutr. 131 (9), 2248–2251. Liang, Y.C., Lin-shiau, S.Y., Chen, C.F., Lin, J.K., 1997. Suppression of extracellular signals and cell proliferation through EGF receptor binding by (-)-epigallocatechin gallate in human A431 epidermoid carcinoma cells. J. Cell Biochem. 67 (1), 55–65. MacDonald, C.J., Ciolino, H.P., Yeh, G.C., 2001. Dibenzoylmethane modulates aryl hydrocarbon receptor function and expression of cytochromes P50 1A1, 1A2, and 1B1. Cancer Res. 61 (10), 3919–3924. Marchand, L.L., Murphy, S.P., Hankin, J.H., Wilkens, L.R., Kolonel, L.N., 2000. J. Natl. Cancer I. 92, 154–160. Mukhtar, H., Ahmad, N., 1999. Green tea in chemoprevention of cancer. Toxicol. Sci. 52 (Suppl. 2), 111–117. Ohishi, T., Kishimoto, Y., Miura, N., et al., 2002. Synergistic effects of (-)-epigallocatechin gallate with sulindac against colon carcinogenesis of rats treated with azoxymethane. Cancer Lett. 177 (1), 49–56. Safe, S., 2001. Molecular biology of the Ah receptor and its role in carcinogenesis. Toxicol. Lett. 120 (1–3), 1–7. Sazuka, M., Imazawa, H., Shoji, Y., Mita, T., Hara, Y., Isemura, M., 1997. Inhibition of collagenases from mouse lung carcinoma cells by
green tea catechins and black tea theaflavins. Biosci. Biotechnol. Biochem. 61 (9), 1504–1506. Schut, H.A., Yao, R., 2000. Tea as a potential chemopreventive agent in PhIP carcinogenesis: Effects of green tea and black tea on PhIPDNA adduct formation in female F-344 rats. Nutr. Cancer 36 (1), 52–58. Schwarz, D., Roots, I., 2003. In vitro assessment of inhibition by natural polyphenols of metabolic activation of procarcinogens by human CYP1A1. Biochem. Biophys. Res. Commun. 303 (3), 902–907. Schwarz, D., Kisselev, P., Roots, I., 2005. CYP1A1 genotype-selective inhibition of benzo[a]pyrene activation by quercetin. Eur. J. Cancer 41, 151–158. Shimizu, Y., Nakatsuru, Y., Ichinose, M., et al., 2000. Benzo[a]pyrene carcinogenicity is lost in mice lacking the aryl hydrocarbon receptor. P. Natl. Acad. Sci. USA 97 (2), 779–782. Steele, V.E., Kelloff, G.J., Balentine, D., et al., 2000. Comparative chemopreventive mechanisms of green tea, black tea and selected polyphenol extracts measured by in vitro bioassays. Carcinogenesis 21 (1), 63–67. Taioli, E., 1999. International collaborative study on genetic susceptibility to environmental carcinogens. Cancer Epidemiol. Biomarkers Prev. 8 (8), 727–728. Wang, H., Wang, Y., Chen, Z.Y., Chan, F.L., Leung, L.K., 2005. Hydroxychalcones exhibit differential effects on XRE transactivation. Toxicology 207 (2), 303–313. Williams, S.N., Shih, H., Guenette, D.K., et al., 2000. Comparative studies on the effects of green tea extracts and individual tea catechins on human CYP1A gene expression. Chem. Biol. Interact. 128 (3), 211– 229. Xu, Y., Ho, C.T., Amin, S.G., Han, C., Chung, F.L., 1992. Inhibition of tobacco-specific nitrosamine-induced lung tumorigenesis in A/J mice by green tea and its major polyphenol as antioxidants. Cancer Res. 52 (14), 3875–3879.
Review
Developing a high-throughput system for the screening of cytochrome P450 1A1 – Inhibitory polyphenols Hau Yi Leung a
a,1
, Yun Wang
b,1
, Ho Yee Chan b, Lai K. Leung
a,b,*
Food and Nutritional Sciences Programme, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong b Department of Biochemistry, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Received 6 January 2007; accepted 17 April 2007 Available online 27 April 2007
Abstract Polycyclic aromatic hydrocarbons (PAH) are established procarcinogens that can be found in our environment. The carcinogenicity of these compounds is initiated by their metabolic intermediates, and the extent of biotransformation determines the amount of reactive intermediates generated. CYP1A1 is a major enzyme that metabolizes PAH into carcinogenic moieties. Since previous studies have shown that increased CYP1A1 activity is associated with a higher cancer risk. Identifying CYP1A1-inhibitory compounds in diet or natural products are of genuine interest for chemoprevention studies. In this project, a stable cell line expressing human CYP1A1 was established for the screening of potential chemopreventive agents. Because of a lacking cellular transport mechanism in assays performed on recombinant protein, an ‘in-cell’ assay system might be a better estimate at the tissue level. Theaflavins were strong inhibitors of ethoxyresorufin-O-deethylase (EROD) activity when assayed on recombinant human CYP1A1 protein. However, this inhibition was not observed in the CYP1A1-expressing cells. The ‘in-cell’ IC50 values determined for compounds such as genistein, quercetin, chalcone, etc. were comparable to the values determined in recombinant protein. This ‘in-cell’ assay has the additional advantages of short sample processing time, and the tedious procedures of protein expression and purification can be waived. 2007 Elsevier Ltd. All rights reserved. Keywords: CYP1A1; Stable transfection; EROD
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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemicals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Cell culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Construction of CYP1A1 expression plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Establishing a stable cell line expressing CYP1A1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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Abbreviations: FBS, fetal bovine serum; AHR, aryl hydrocarbon receptor; XRE, xenobiotic response element; DMBA, 7,12-dimethylbenz[a]anthracene; PAH, polycylic aromatic hydrocarbon; B[a]P, benzo[a]pyrene; EROD, ethoxyresorufin-O-deethylase; DMSO, dimethyl sulfoxide; EGCG, epigallocatechin gallate. * Corresponding author. Present address: Department of Biochemistry, The Chinese University of Hong Kong, Rm. 507C MMW Bldg, Shatin, N.T., Hong Kong. Tel.: +852 26098137; fax: +852 26037732. E-mail address: [email protected] (L.K. Leung). 1 These authors contributed equally to this work. 0887-2333/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2007.04.005
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3.1. Enzyme inhibition assays performing in intact MCF-7 cells expressing CYP1A1 . . . . . . . . . . . . 3.2. Enzyme inhibition assays performing in recombinant CYP1A1 . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Enzyme kinetic studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Statistical methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. EROD activity inhibited by polyphenols in cells over-expressing CYP1A1 . . . . . . . . . . . . . . . . . 4.2. Enzyme kinetic study of chalcone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Effects of selected tea polyphenols on EROD activity in recombinant CYP1A1 protein and cells Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
2. Materials and methods
Polycylic aromatic hydrocarbons (PAH) are common toxicants in our environment. They can be extracted from diesel exhaust, char broiled meat, tobacco smoke, overheated cooking oil, etc. (Environmental Protection Agency, 1990; International Agency for Research on Cancer, 1983). PAHs are classified as procarcinogens because these compounds are ultimately metabolized into DNA-attacking electrophiles in our body. Aryl hydrocarbon receptor (AHR) reconciles the transformation of these procarcinogens into genotoxic moieties in a series of reactions. Upon PAH activation, the AHR complex translocates from cytosol to the nucleus and interacts with an AHR nuclear translocator (ARNT). This protein complex docks onto a gene containing xenobiotic responsive elements (XRE) and initiates its transcription (Kronenberg et al., 2000). Cytochrome P450 1A1 (CYP1A1) is one of these downstream genes of AHR transactivation (Dertinger et al., 2000; Safe, 2001). The subsequent translated CYP1A1 enzyme biotransforms PAHs into DNA-attacking species. The observation that benzo[a]pyrene failed to induce CYP1A1 and liver cancer in AHR-knockout mice has illustrated this inter-relationship (Shimizu et al., 2000). Studies have shown that polymorphisms with higher activity of CYP1A1 are associated with increased risk of various cancers (Taioli, 1999). In contrast, the inhibition of CYP1 enzymes appears to be beneficial in the prevention of 7,12-dimethylbenz[a]anthracene (DMBA)–DNA adduct formation in vivo and in vitro (Kleiner et al., 2002; MacDonald et al., 2001). Developing a fast screening methodology for CYP1A1-inhibitory compounds is prompted by these findings. Previously we employed an XRE-driven reporter assay technique to assess CYP1 expression and found that the luciferase activity suppressed by polyphenols correlated to the reduced DMBA–DNA adduct formation (Chan and Leung, 2003). To complement the reporter assay, the present study aimed to establish a stable cell line for the screening of CYP1A1 inhibitor at the enzyme level.
2.1. Chemicals
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7,12-Dimethylbenz[a]anthracene (DMBA) and DMSO were obtained from Sigma–Aldrich Fine Chemicals (St Louis, MO, USA). Theaflavins were isolated as previously described (Leung et al., 2001). All other chemicals, if not stated, were acquired from Sigma Chem. 2.2. Cell culture MCF-7 cells (obtained from American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI – 1640 phenol red free media (Sigma–Aldrich) and 10% fetal bovine serum (Invitrogen Life Technology, Rockville, MD, USA) at 37 C and 5% carbon dioxide. 2.3. Construction of CYP1A1 expression plasmid Total RNA was isolated from cells grown in six-well plates (Costar). RNA of 1 lg was used for the first strand cDNA synthesis, and the final volume was diluted to 20 ll. Primers encompassed the full length of CYP1A1 were designed and a Perkin Elmer Thermocycler (GeneAmp PCR System 2400) was utilized to amplify the target cDNA. A PCR reaction consisted of 0.2 mmol/l dNTP, 2 ll cDNA, 0.2 lmol/l of each primer, 1 · PCR buffer and 1 U of pfu polymerase (Invitrogen). The conditions were 94 C for 45 s, 65 C for 45 s, 72 C for 1 min and a final extension period of 7 min at 72 C for 25 cycles. The PCR product was separated on a 1.8% agarose gel, stained with ethidium bromide, and extracted into a 1.5 ml eppendorf tube for enzyme digestion. The digested fragment was inserted into the mammalian expression vector pcDNA3.1 (Promega) and the cDNA sequence of hCYP1A1 was confirmed subsequently. 3. Establishing a stable cell line expressing CYP1A1 The expression construct was transfected into MCF-7 cells by lipofectamine (Invitrogen) and selected by geneticin
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activities were expressed as a relative EROD activity to the control. 3.2. Enzyme inhibition assays performing in recombinant CYP1A1 Recombinant CYP1A1 proteins expressed in microsomes (Supersomes) were purchased from BD Gentest Corp. (Woburn, MA, USA). Chalcone and tea polyphenols were tested using recombinant CYP1A1 for comparison. Protein of 2 pmol was incubated in 100 ll PBS, pH 7.2 with 400 nM ethoxyresorufin and polyphenol in different concentrations. The reaction was initiated by 500 lM NADPH, and terminated by 100 ll of ice-cold methanol after 20 min of incubation. The fluorescence was measured as described earlier. Fig. 1. Establishing a stable cell line expressing CYP1A1 MCF-7 cells were transfected with CYP1A1 expression vector and selected. RT-PCR was used to analyze the expression of clones A1–A12 as shown in Fig. 1a. EROD activity of the clones was also performed to verify the mRNA expression (Fig. 1b).
(Invitrogen). After maintaining the transfected cells for 21 days, clones were examined for the message expression and activity by RT-PCR (Fig. 1a) and ethoxyresorufin-Odeethylase (EROD) activity (Fig. 1b). Clones that consistently had the highest expression and activity were selected for the establishment of a stable CYP1A1 expressing cell line. Clones A5 and A10 displayed a distinct expression of CYP1A1 in RT-PCR, and subsequent EROD assay confirmed that Clone A10 had an enzyme activity 14 times of those clones transfected with empty vector. Contrasting to a 2-fold increase in cells treated with DMBA (Chan et al., 2003b), these Clone A10 cells designated as MCF-7CYP1A1 provided an excellent alternative to xenobiotic-induced model for the screening of CYP1A1-inhibitory compounds. 3.1. Enzyme inhibition assays performing in intact MCF-7 cells expressing CYP1A1 The assay method was performed as previously described (Chan et al., 2003a) with some modifications. In brief, MCF-7CYP1A1 cells were seeded in 96-well plates for 24 h. The medium was then removed and EROD activity assays were then carried out. Ethoxyresorufin (5 lM) and salicylamide (1.5 mM) of 50 ll were dissolved in PBS, and tested phytochemicals were added in wells and incubated at 37 C for 15 min. The reactions were stopped by 50 ll of ice-cold methanol, and the resorufin generated was measured by a FLUOstar Galaxy microplate reader (BMG Labtechnologies, Offenburg, Germany) with 544 nm and 590 nm as the respective excitation and emission wavelengths. The activities were quantified against resorufin standards. In order to avoid a batch-to-batch variation in cell number and viability, timing, etc., the
3.3. Enzyme kinetic studies All the above conditions, described in assays performing in cell or recombinant protein, were applied except that many substrate concentrations were tested. 3.4. Statistical methods A SigmaPlot software package (IL, USA) was utilized for statistical analysis. The results, whenever applicable, were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparison test if significant differences (p < 0.05) were observed. T-test was performed for the other comparisons. For the IC50 estimations, the non-linear curve-fitting program installed in the software was employed. 4. Results 4.1. EROD activity inhibited by polyphenols in cells overexpressing CYP1A1 Some flavonoids were tested on the CYP1A1 inhibition in the MCF-7CYP1A1 cells (Fig. 2). The inhibitory results of chalcones, isoflavones, flavonols and other phytochemicals on EROD activity are shown in Fig. 2a–c, and the estimated IC50 are listed in Table 1. Among all types of flavonoids tested, the flavonol quercetin and kaempferol displayed the strongest inhibitory effect on CYP1A1. In the isoflavone category, baicalein was the most potent, followed by genistein, equol and biochanin A. Daidzein did not inhibit the EROD activity in the current study. 2 0 OH and 2-OH chalcones had the lowest IC50 values, whereas butein was the weakest. 4.2. Enzyme kinetic study of chalcone Enzyme kinetic assays on the polyphenol chalcone were carried out in MCF-7CYP1A1 cells and recombinant protein for comparison. The Ki’s (Fig. 3b and e), which were
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Fig. 2. Inhibitory action of polyphenols on CYP1A1 in MCF-7CYP1A1 cells. The inhibitory effects of chalcones, isoflavones and flavonols and other phytochemicals were determined in CYP1A1 stable cells as shown in Fig. 2a, 2b and 2c, respectively. Values are means ± S.D., n = 3. Table 1 Estimated IC50 values from MCF-7CYP1A1 ‘in-cell’ assays Flavonoids Chalcones: 2 0 -OH chalcone 2-OH chalcone 4-OH chalcone 4,2 0 ,4 0 -trihydroxychalcone Butein Isoflavones: Equol Daidzein Genistein Biochanin A Baicalein
Estimated IC50 (lM) 11.14 8.64 16.14 13.85 25.00 >44.44 50.00 33.56 24.44 10.43
Flavonols: Quercetin Kaempferol
1.36 5.43
Others: Luteolin Resveratrol Naringenin
6.74 36.11 >100.00
recombinant protein assay. The ‘in-cell’ assaying method could be applied for enzyme kinetic analysis.
Estimated ki (lM) 0.38 0.12 4.10 5.20 8.50 – – 15.35 4.00 0.40 0.20 0.30
4.3. Effects of selected tea polyphenols on EROD activity in recombinant CYP1A1 protein and cells EROD activity was determined in intact MCF-7CYP1A1 cells and in recombinant protein (Supersomes BD Gentest, Wobash, MA). Some tea polyphenols displayed strong inhibition on EROD activity assayed in Supersomes. The IC50 values were estimated to be 65 lM for theaflavin 1 (TF1), theaflavin 3 0 -gallate (TF2) and theaflavin-3,3 0 -digallate (TF3), and about 30 lM for epigallocatechin gallate (EGCG) (Fig. 4a). However, the EROD activity was neither significantly suppressed in DMBA-induced cells (Fig. 4b) nor in MCF-7CYP1A1 cells (Fig. 4c). 5. Discussion
0.50 11.40 –
derived from the Lineweaver–Burk plot (Fig. 3a and c), were determined to be 0.5 lM in-cell assay and 1.4 lM in
With regard to the CYP1A1-inhibitory activities performed in stable cells, the trend of data was consistent with that performed in recombinant protein as we previously reported (Chan and Leung, 2003; Wang et al., 2005). The estimated IC50 values of reverstrol, quercetin, kaempferol and naringenin in the present study are greater than the
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Fig. 3. ‘In-cell’ enzyme kinetic determination EROD assays were performed on MCF-7CYP1A1 cells treated with 0, 0.5, 1, 5 and 10 lM of chalcone. The Lineweaver–Burk plot (Fig. 3a) was generated from the reciprocal data, and the value of Ki was derived from the slopes of the regression lines (Fig. 3b). For comparison, Fig. 3c and 3d are Lineweaver–Burk plot and Ki derivation from data obtained from recombinant protein. Values shown are mean ± S.D., n = 3.
values obtained from recombinant protein assays (Schwarz and Roots, 2003) by 2- to 20-fold. Although the ‘in-cell’ assay tends to over-estimate the IC50 values, the method can still be a viable substitution for recombinant protein. The differences, which could be attributed to the cellular transport or metabolism of the administered chemicals, would be a better predictor at the physiological level. Among the green tea catechins, (–)-epigallocatechin gallate (EGCG) has stronger and higher anti-proliferative, anti-neoplastic and anti-mutagenic activities than (–)-epicatechin (EC) and (–)-epicatechin gallate (EGC) (Ahmad and Mukhtar, 1999; Liang et al., 1997; Mukhtar and Ahmad, 1999). Oral or topical administration of EGCG inhibits 4-(methylnitrosamino)-1-(3-pyridyl)-1butanone (NNK)-induced lung tumors (Xu et al., 1992), azoxymethane (AOM)-induced colon cancer (Ohishi et al., 2002), and DMBA-induced breast cancer (Kavanagh et al., 2001). These studies indicate that EGCG is protective against chemical-induced carcinogenesis. Other studies have demonstrated that EGCG can inhibit the biotransformation of carcinogens by modulating CYP1A enzyme expression and activities, and decreasing the production of ultimate carcinogens (Williams et al., 2000; Xu et al., 1992). However, our results performed on MCF-
7CYP1A1 cells suggested that EGCG was merely a weak inhibitor on CYP1A1. Theaflavins also exhibit anti-carcinogenesis effects in different models, and little is known about the mechanism of its chemoprevention. They inhibit neoplastic transformation in human lung cells (Sazuka et al., 1997), mammary cells and rat tracheal epithelial cells (Steele et al., 2000). Similar to green tea polyphenols, they are strong antioxidants (Leung et al., 2001) and are capable of inhibiting DNA-carcinogen binding (Schut and Yao, 2000). Others have suggested that theaflavins may play a role in modulating carcinogens activation (Apostolides et al., 1997). Black tea has been demonstrated to be inhibitory in benzo[a]pyrene (B[a]P) metabolism in a cell-free system (Hammons et al., 1999) and in a hepatocarcinoma cell culture model (Feng et al., 2002). Our screening results also indicated that theaflavins displayed minimal CYP1A1-inhibition at the cellular level. Since theaflavins have high molecular weight, they might not be able to reach the vicinity in interacting with the enzyme. In a recent case–control study quercetin consumption has been associated with reduced lung cancer risk in Hawaii (Marchand et al., 2000). Compared to an IC50 value of 0.2 lM determined in recombinant protein
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Fig. 4. Differential inhibitory effects of tea polyphenols on EROD activity in recombinant CYP1A1 protein and cells CYP1A1 inhibitory effects of tea polyphenols – epigallocatechin gallate (EGCG), theaflavin 1 (TF1), theaflavin-3,3 0 -digallate (TF2) and theaflavin 3 0 -gallate (TF3) – were assayed in different systems. Fig. 4a represents the CYP1A1-inhibitory results of some tea polyphenols performed in recombinant protein. Fig. 4b illustrates the inhibition assay carried out in DMBA-induced MCF-7 cells. Fig. 4c indicates the ‘in-cell’ assay performed in MCF-7CYP1A1 cells. Values are means ± SD, n = 3.
(Schwarz et al., 2005), the value of quercetin was determined to be 1.36 lM in the present study. Quercetin was the most effective suppressor of CYP1A1 among various dietary chemicals tested in the present study. In addition quercetin is an agonist of AHR (Ciolino et al., 1999), it may compete for the binding of AHR and reduce the expressions of CYP1A1. Quercetin appears to be a potent dietary phytochemical in the protection against PAH toxicity. In summary, this study indicated that CYP1A1 stable cell line could be a replacement for the recombinant protein system. Because the assays can be performed in 96-well plates and no protein purification is required, this stable cell line is a better alternative for the screening of dietary CYP1A1-inhibitory compounds. References Ahmad, N., Mukhtar, H., 1999. Green tea polyphenols and cancer: Biologic mechanisms and practical implications. Nutr. Rev. 57 (3), 78– 83. Apostolides, Z., Balentine, D.A., Harbowy, M.E., Hara, Y., Weisburger, J.H., 1997. Inhibition of PhIP mutagenicity by catechins, and by theaflavins and gallate esters. Mutat. Res. 389 (2–3), 167–172. Chan, H.Y., Leung, L.K., 2003. A potential protective mechanism of soya isoflavones against 7,12-dimethylbenz[a]anthracene tumour initiation. Br. J. Nutr. 90 (2), 457–465.
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