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Carcinogenesis Advance Access originally published online on October 29, 2005
Carcinogenesis 2006 27(3):656-663; doi:10.1093/carcin/bgi256
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Carcinogenesis vol.27 no.3 © Oxford University Press 2005; all rights reserved.

Potent inhibition of carcinogen-bioactivating cytochrome P450 1B1 by the p53 inhibitor pifithrin {alpha}

Lydie Sparfel *, Julien Van Grevenynghe, Marc Le Vee, Caroline Aninat and Olivier Fardel

INSERM U620, IFR 140, Université de Rennes I, 2 Avenue du Pr Léon Bernard, 35043 Rennes, France

* To whom correspondence should be addressed. Tel: +33 2 23 23 48 68; Fax: +33 2 23 23 47 94; Email: lydie.sparfel{at}rennes.inserm.fr


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Pifithrin {alpha} (PFT{alpha}) is a chemical compound that inhibits p53-mediated gene activation and apoptosis. It has also been recently shown to alter metabolism of carcinogenic polycyclic aromatic hydrocarbons (PAHs). This has led us to examine the effect of PFT{alpha} on the activity of cytochrome P-450 (CYP) 1 isoforms, known to metabolize PAHs, such as benzo(a)pyrene (BP), into mutagenic metabolites. We report that PFT{alpha} caused a potent inhibition of CYP1-related activity as measured by ethoxyresorufin O-deethylase activity in CYP1-containing MCF-7 cells and liver microsomes. It also directly affected the catalytic activity of human recombinant CYP1A1, CYP1A2 and CYP1B1 isoforms, with a potent inhibitory effect towards CYP1B1. The nature of this CYP1B1 inhibition by PFT{alpha} was mixed-type with an apparent Ki of 4.38 nM. Blockage of CYP1 activity by PFT{alpha} was associated with a decreased metabolism of BP, a reduced formation of BP-derived adducts and a diminished BP-induced apoptosis in human cultured cells targets for PAHs like primary human macrophages and p53-negative KG1a leukaemia cells. These data further substantiate an unexpected and p53-independent action of PFT{alpha} for preventing toxicity of chemical carcinogens such as PAHs, through inhibition of CYP1 enzyme activities, especially that of CYP1B1.

Abbreviations: AhR, aryl hydrocarbon receptor; BP, benzo(a)pyrene; CYP, cytochrome P-450; PFT{alpha}, pifithrin {alpha}; EROD, ethoxyresorufin O-deethylase; MC, 3-methylcholanthrene; PAH, polycyclic aromatic hydrocarbon; TCDD, 2,3,5,7-tetrachlorodibenzo-p-dioxin


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (BP), are ubiquitous environmental contaminants generated by a variety of industrial processes (1). They exert a wide range of toxic effects such as carcinogenic and apoptotic effects. The molecular mechanisms responsible for these effects have been linked to the aryl hydrocarbon receptor (AhR), an ubiquitously expressed cytosolic protein that is a member of the basic helix–loop–helix superfamily of transcriptional factors. Activation of AhR involves PAH-binding and dissociation with the molecular chaperone heat shock proteins, thereby, triggering its translocation in the nucleus where it dimerizes with the AhR nuclear translocator, followed by interaction with xenobiotic responsive elements found in 5'-flanking regions of responsive genes (2). Among these genes are those encoding cytochromes P450 (CYPs) 1A1, 1A2 and 1B1 enzymes. Interestingly, PAHs are not only agonists of the AhR but also substrates for the induced CYP1A1, CYP1A2 and CYP1B1 enzymes, which metabolize them into reactive intermediates that can covalently bind DNA to form mutagenic DNA adducts, and hence might be involved in the initial events of carcinogenesis (3).

The chemical compound, pifithrin {alpha} (PFT{alpha}), was originally thought to be a specific inhibitor of the tumour suppressor protein p53 signalling (4). In this context, PFT{alpha} has been successfully used in vitro and in vivo to protect normal cells from lethal genotoxic stress caused by gamma radiation and chemotherapy, suggesting a possible clinical use of PFT{alpha} to reduce side effects that occur during antitumour therapy (5). In addition, PFT{alpha} is considered a useful tool in the laboratory to characterize p53-mediated events using a variety of cell types and apoptotic inducing agents, including PAHs (6,7). More recently, PFT{alpha} has been shown to reduce BP metabolism in intact Hepa1c1c7 cells, as measured by the generation of tetrols and by covalent binding of BP to macromolecules, resulting in an inhibition of BP-induced apoptosis and a direct effect of PFT{alpha} on BP-activation via the CYP1A1 was suggested (8). To test such a hypothesis in the present study, we have analysed the effects of PFT{alpha} both on CYP1 activity and BP-mediated toxicity in various experimental models. PFT{alpha} was found to reduce the activity of different CYP1 isoforms, namely, CYP1A1, CYP1A2 and especially CYP1B1, thereby, preventing BP bioactivation and toxicity in human cells targets for PAHs such as primary macrophages and leukaemic KG1a cells. Such data further clarify a new mechanism of action of PFT{alpha}, which is likely to contribute to its protective effects towards chemical carcinogens like PAHs.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Chemicals and reagents
BP, 3-methylcholanthrene (MC) and ethoxyresorufin were supplied from Sigma Chemicals Co. (St Louis, MO) and PFT{alpha} was from Calbiochem (La Jolla, CA). 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was provided by Cambridge Isotopes Laboratories whereas [3H]BP (specific activity 50 Ci/mmol) was purchased from Isobio (Fleurus, Belgium). Microsomes from human B lymphoblastoid cells co-expressing NADPH-CYP reductase and human recombinant CYP1A1, CYP1A2 or CYP1B1 were purchased from Gentest (Woburn, MA). All other compounds were commercial products of the highest purity available.

Cell isolation and culture
Peripheral blood mononuclear cells were obtained from blood buffy coats (kindly provided by Etablissement Français du Sang, Rennes, France) through Ficoll (Life Technologies, Cergy-Pontoise, France) gradient centrifugation. After a selective 2 h step of adhesion, human adherent monocytes were cultured for 6 days in RPMI 1640 medium (Eurobio, Les Ulis, France), supplemented with 2 mM L-glutamine, 20 UI/ml penicillin, 20 µg/ml streptomycin and 10% fetal calf serum (In Vitrogen, NY) in the presence of 400 U/ml human GM-CSF (specific activity 1,2 x 108 U/mg) (Schering Plough, Lyon, France). As described previously (9), such a protocol permits to obtain pure macrophage cultures with <1% of contaminating cells.

The human leukaemic cell line KG1a and the human mammary tumour cell line MCF-7 were both cultivated in RPMI 1640 medium supplemented with 2 mM L-glutamine, 20 UI/ml penicillin, 20 µg/ml streptomycin and 10% fetal calf serum.

Chemicals were used as stock solutions in dimethylsulfoxide; the final concentration of this solvent in culture medium was always <0.2% (v/v) and control cultures received the same dose of vehicle as treated cultures.

RNA isolation and RT–PCR assay
Total RNA was isolated from cells using the TRIzol method (Life Technologies). Total RNA (2 µg) was first reverse-transcribed using the Superscript II reverse transcriptase protocols (Life Technologies) and equal aliquots of cDNA were subsequently amplified by using the PCR Master Mix from Promega (Madison, WI). The gene-specific primers used were as follows: CYP1A2 sense, 5'-CTTTGACAAGAACAGTGTCCG-3'; CYP1A2 antisense, 5'-AGTGTCCAGCTCCTTCTGGAT-3', CYP1B1 sense, 5'-AAAGAGGTACAACATCACCT-3'; CYP1B1 antisense, 5'-GTATATTGTTGAAGAGACAG-3' and GAPDH sense, 5'-TTCACCACCATGGAGAAGGC-3'; GAPDH antisense, 5'-GGCATGGACTGTGGTCATGA-3'. The primers used for CYP1A1 detection were exactly those described by van Grevenynghe (9). Analysis of GAPDH mRNA levels, not affected by TCDD or BP, was routinely performed as a control. PCR analyses were carried out from the logarithmic phase of amplification. PCR products were separated on 1% agarose gels and stained with ethidium bromide.

Preparation of total cell lysates
Total cellular protein extracts were obtained by incubating human macrophages and KG1a cells in a lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1 mM EGTA, 0.1% Tween-20, 10% glycerol, 100 µM phenylmethanesulphonyl fluoride, 10 mM dithiothreitol, 2 µg/ml leupeptin and 1 µg/ml pepstatin as described previously (9) and then stored at –20°C. Protein contents were determined using the Bradford's dosage (10).

Rat treatment
Male Wistar rats weighing 200–250 g were treated with the PAH MC (20 mg/kg/daily i.p.) dissolved in oil for 5 days. All procedures were in accordance with the regulations laid down by the French Ministry of Agriculture and Forest, for the care and use of laboratory animals. At the end of the treatment, rats were killed and livers were immediately removed and kept at –80°C until use.

Preparation of microsomal fractions
Microsomal fractions were prepared from rat livers by differential centrifugation in 50 mM Tris–HCl, pH 7.4, containing 0.25 M sucrose and 1 mM EDTA as described previously (11); they were stored at –80°C in 0.1 M phosphate-buffered saline pH 7.4, containing 10% glycerol. Protein contents were determined using the Bradford's dosage (10).

Western blotting immunoassays
Proteins from total cell lysates and microsomal proteins were separated on a polyacrylamide gel and electrophoretically transferred onto nitrocellulose membranes (Bio-Rad, Marne la Coquette, France). After blocking, membranes were incubated with a goat anti-human CYP1A1/2 antibody (Daiichi Pure Chemicals Co, Tokyo, Japan) or with a mouse anti-human p53 antibody (Dakocytomation, Trappes, France) raised against total p53 protein. A peroxidase-conjugated antibody was next used as secondary antibody and blots were developed by chemoluminescence using the Amersham ECL detection system (Amersham, Orsay, France).

CYP activities in cultured cells and microsomes
Ethoxyresorufin O-deethylase (EROD) activity was used as a measurement of CYP1A1, CYP1A2 and CYP1B1 activities (12,13) in TCDD-exposed MCF-7 cells, microsomal fractions and human recombinant CYP1 isoforms in the presence of various concentrations of PFT{alpha} (0–20 µM). Resorufin formation was monitored using a SpectraMax Gemini spectrofluorimeter (Molecular Devices, Sunnyvale, CA); excitation and emission wavelengths were 544 and 590 nm, respectively. Reaction rates were determined under linear conditions with various incubation times and protein concentrations.

To characterize the potential inhibitory effects of PFT{alpha} towards CYP1B1-mediated EROD activity, EROD assay was conducted at ethoxyresorufin concentrations ranging from 2.5 to 20 µM, in the presence of various PFT{alpha} concentrations (0–160 nM), using recombinant CYP1B1-containing microsomes. Kinetic enzymatic parameters were estimated using a computer program designed for non-linear regression analysis (GraphPad Software, Prism 3.02), according to the Michaelis–Menten equation. The apparent Michaelis–Menten constant (Km) and the maximal velocity (Vmax) were obtained by non-linear regression analysis of enzymatic velocity versus different PFT{alpha} concentrations. Inhibition constant (Ki) was determined from secondary plot of Km/Vmax versus 1/[PFT{alpha}]. To determine the mode of inhibition, Lineweaver–Burk linear regression was used for graphic plot of 1/V versus 1/[ethoxyresorufin].

BP metabolism
Primary human macrophages, previously exposed to 10 nM TCDD for 24 h in order to induce CYP1A1/1B1 expression (14), were treated by 0.1 µg/ml of [3H]BP in the absence or presence of 20 µM PFT{alpha} for 4 h. Water-soluble BP metabolites were then extracted and quantified as described previously (11) by scintillation counting. Spontaneous cell-independent conversion of BP into water-soluble metabolites was detected in parallel by incubating [3H]BP into medium in the absence of cells.

Measurement of BP-derived adducts
KG1a cells, previously exposed to 10 nM TCDD for 24 h in order to induce CYP1A1/1B1 expression (14), were treated by 0.1 µg/ml [3H]BP in the absence or presence of 20 µM PFT{alpha} for 4 h. After two phosphate-buffered saline washes, proteins and nucleic acids were extracted using a trichloroacetic acid precipitation method (15). Amount of tritiated BP metabolites covalently bound to cellular nucleophile macromolecules were then determined by scintillation counting and normalized to amounts of total proteins quantified by Bradford's dosage (10).

Detection of apoptosis
Human macrophages and KG1a cells were treated by 1 µM BP in the absence or presence of 20 µM PFT{alpha}. Light microscopic detection of apoptosis was determined in BP-treated macrophages using Hoechst labelling as described previously (11), whereas assessment of apoptosis was performed in BP-treated KG1a cells using the FITC-conjugated annexinV Kit (Beckman Coulter, Marseille, France) for detection of externalized phosphatidylserine membrane residues of apoptotic cells. Briefly, after washing in phosphate-buffered saline at 4°C, cells were incubated with 2 µl FITC-annexinV and 5 µl propidium iodide in Ca2+ binding buffer for 10 min at 4°C and were finally analysed by flow cytometry using a FACScalibur cytometer (Becton Dickinson, San Jose, CA).

Statistical analysis
Data were analysed using the paired Student's t-test or the non-parametric Wilcoxon's test. For evaluation of the variations of EROD activity measured in the absence or presence of various concentrations of PFT{alpha}, analysis of variance followed by a multirange Dunnett's t-test was used.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
PFT{alpha} decreases CYP1-related EROD activity in TCDD-treated MCF-7 cells without down-modulating CYP1-mRNA and protein levels
To investigate the effect of PFT{alpha} treatment on catalytic activities of CYP1 enzymes, we used the EROD assay, which is specific for the CYP1 family (12,13). The activity was measured in MCF-7 cells previously exposed to 10 nM TCDD for 24 h in the presence of increasing concentrations of PFT{alpha} (0–20 µM), which did not exert any cytotoxic effects (data not shown). Figure 1A shows that PFT{alpha} significantly decreased TCDD-induced EROD activity in MCF-7 cells. This may result from either direct inhibition of CYP1 catalytic activity or, alternatively, from alteration of CYP1 expression. To investigate this latter point, the effects of PFT{alpha} on CYP1 expression were then examined at both mRNA and protein levels. RT–PCR was first used to analyse mRNA levels of the different CYP1 isoforms, i.e. CYP1A1, CYP1A2 and CYP1B1 in MCF-7 cell line. As shown in Figure 1B, basal CYP1A1 and CYP1A2 mRNA levels were very low in control MCF-7 cells, whereas the human CYP1B1 gene was expressed at substantial levels; all of these CYP1 isoforms were markedly induced by a 24 h treatment by 10 nM TCDD. However, PFT{alpha} treatment did not modify TCDD-induced CYP1A1, CYP1A2 and CYP1B1 mRNA levels. Likewise, it failed to alter upregulation of CYP1A1/2 protein amounts in TCDD-treated MCF-7 cells as assessed by western blotting (Figure 1C). Interestingly, exposure to PFT{alpha} alone resulted in a moderate induction of CYP1 mRNA and protein levels in MCF-7 cells (Figures 1B and C), which is fully in agreement with the fact that PFT{alpha} has been very recently reported first to induce CYP1A1 expression (8), and next, to bind to AhR and to induce formation of its DNA-binding complex, thus upregulating CYP1A1 via AhR (16).


Figure 1
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  		Fig. 1. Effects of PFT{alpha} on EROD activity (A) and CYP1 mRNA (B) and protein (C) levels in human mammary tumour MCF-7 cells. (A) MCF-7 cells were treated by 10 nM TCDD in the absence or presence of increasing concentrations of PFT{alpha} (0–20 µM) for 24 h. EROD activity was then determined as described in ‘Materials and methods’. Data are expressed as the percentages of control EROD values measured in TCDD-treated cells not exposed to PFT{alpha} (44.40 ± 12.01 pmol resorufin/min/mg proteins) and are the means ± S.D. of three independent experiments performed in triplicate. **indicates P < 0.01, when compared with PFT{alpha}-untreated cells. (B) and (C) MCF-7 cells were either untreated (UNT), treated by 10 nM TCDD or 20 µM PFT or co-exposed to TCDD and PFT{alpha} for 24 h and levels of CYP1 mRNA (B) and protein (C) were determined by RT–PCR and western blotting, respectively. Data are representative of three independent experiments.

 
PFT{alpha} directly inhibits catalytic activity of CYP1 isoforms, especially that of CYP1B1
PFT{alpha} was further examined for its ability to directly inhibit CYP1-dependent EROD activity. Significant and dose-dependent inhibition of EROD activity by PFT{alpha} was found in isolated microsomes from MC-treated rat livers (Table I). To investigate whether PFT{alpha} affected the catalytic activity of different human isoforms of the CYP1 family, namely, CYP1A1, CYP1A2 and CYP1B1, the EROD assay was then conducted with microsomes containing the different corresponding recombinant CYP1 enzymes in the presence of various concentrations of PFT{alpha}. As shown in Figure 2, PFT{alpha} decreased CYP1A1, CYP1A2 and CYP1B1 catalytic activities in a concentration-dependent manner. PFT{alpha} showed a potent inhibitory effect on CYP1B1 activity (IC50 = 20.63 nM) and, to a lesser extent, on CYP1A1 (IC50 = 1.53 µM) and CYP1A2 (IC50 = 0.77 µM) activities.


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  		Table I. Effects of increasing concentrations of PFT{alpha} (0–20 µM) on EROD activity in isolated microsomes from MC-treated rat livers

 

Figure 2
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  		Fig. 2. Effects of PFT{alpha} on EROD activity in recombinant human CYP1A1-, CYP1A2-containing (A) and CYP1B1- (B) containing microsomes. CYP1A1-, CYP1A2- and CYP1B1-related EROD activities were determined in the presence of various increasing concentrations of PFT{alpha} as described in ‘Materials and methods’. Data are expressed as the percentages of control EROD values measured in the absence of PFT{alpha} (86.89 ± 3.28 pmol resorufin/min/mg protein for CYP1A1, 20.40 ± 3.16 pmol resorufin/min/mg protein for CYP1A2, 16.77 ± 4.23 pmol resorufin/min/mg protein for CYP1B1) and represent the mean of three independent experiments performed in duplicate. **indicates P < 0.01, when compared with PFT{alpha}-untreated cells.

 
To further characterize the inhibition of CYP1B1 catalytic activity by PFT{alpha}, enzyme kinetic experiments were performed with different substrate concentrations (2.5–20 µM) in the presence of increasing PFT{alpha} concentrations (0–160 nM) in microsomes containing recombinant CYP1B1. Lineweaver-Burk plot of enzyme kinetic data were consistent with a mixed-type inhibitory effect of PFT{alpha} with an apparent Ki of 4.38 nM (Figure 3).


Figure 3
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  		Fig. 3. Inhibition kinetic of human CYP1B1-mediated EROD activity by PFT{alpha}. Recombinant CYP1B1-containing microsomes were incubated with increasing concentrations of ethoxyresorufin (2.5–20 µM) in the presence of increasing PFT{alpha} concentrations (0–160 nM) and EROD activity was determined as described in ‘Materials and methods’. (A) Representation of Lineweaver-Burk linear regression was generated. (B) Plot of Km/Vmax versus PFT{alpha} concentrations was obtained from Michaelis–Menten non-linear regression; apparent Ki calculated as the negative value of x-axis intercept was 4.38 nM. Data are generated from one representative out of two independent experiments performed in duplicate.

 
PFT{alpha} inhibits CYP1-dependent BP toxicity in primary human macrophage cultures and in the p53-negative KG1a cell line
PAHs are procarcinogens that require metabolic activation through the action of CYP, especially CYP1A1/1B1, to exert their deleterious effects (3). To further explore the effects of PFT{alpha} on CYP1 activity-related cellular processes, we examined the effects of PFT{alpha} on the formation of BP-derived metabolites and on BP toxicity in primary human macrophages shown previously to be targets for PAHs (17). Cell-linked formation of BP-derived metabolites was found to be hugely inhibited by addition of 20 µM PFT{alpha} (Figure 4A), which, moreover, markedly reduced BP-induced apoptosis in human macrophages (Figure 4B). Such a protective effect of PFT{alpha} towards PAH-induced apoptosis has also been recently described in MCF-7 cells (18). Interestingly, although PFT{alpha} has been previously reported to inhibit apoptosis in response to DNA damage through inhibition of p53 translocation from cytoplasm to nucleus without obviously preventing p53 upregulation (4), it was found to completely inhibit upregulation of p53 expression occurring in BP-treated macrophages (Figure 4C) and most likely reflecting BP-induced DNA damage as described previously (19). Since previous studies have shown that PFT{alpha} also inhibits p53 upregulation in BP-treated Hepa1c1c7 cells (20), and have an effect on BP metabolism (8), these results further substantiate that PFT{alpha} may primarily block apoptosis through inhibiting BP metabolite formation and subsequent BP metabolites-triggered induction of p53 instead of counteracting p53 signalling. To validate this hypothesis, we then used the p53-negative KG1a cell line. BP-triggered apoptosis was found to be nearly fully blocked by PFT{alpha} in KG1a cells (Figure 5A). As already described (21), these cells, however, failed to exhibit detectable levels of p53, even after exposure to BP (Figure 5B). By contrast, BP-treated KG1a cells showed upregulation of metabolizing enzymes such as CYP1A1 and CYP1B1 (Figure 5C), which are probably fully active as assessed by the formation of BP-derived adducts (Figure 5D). This formation of BP metabolites-derived adducts was blocked by PFT{alpha} (Figure 5D), suggesting that its protecting effects towards apoptosis was due to inhibition of BP metabolite formation in this p53-negative cell line.


Figure 4
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  		Fig. 4. Effects of PFT{alpha} on BP metabolism (A), BP-induced apoptosis (B) and p53 expression (C) in human macrophages. (A) TCDD-pretreated human macrophages were exposed to 0.1 µg/ml [3H]BP for 4 h in the absence or presence of 20 µM PFT{alpha}. The amount of water-soluble [3H]BP metabolites generated was then measured by scintillation counting as described in ‘Materials and methods’. Spontaneous cell-independent conversion of BP into water-soluble metabolites was detected in parallel by incubating [3H]BP into medium in the absence of cells. The results are expressed as c.p.m./well and are the means ± S.D. of three independent experiments performed in duplicate. ***indicates P < 0.001, when compared with PFT{alpha}-untreated cells. (B) and (C) Human macrophages were either untreated (UNT), treated by 1 µM BP or 20 µM PFT{alpha} or co-exposed to BP and PFT{alpha} for 7 days. (B) Apoptotic cells were detected by fluorescence microscopy after Hoechst 33342 staining of nuclei as described in ‘Materials and methods’. Data are expressed as the percentages of apoptotic cells and are the means ± S.D. of five independent experiments. ***indicates P < 0.001, when compared with BP-treated cells not exposed to PFT{alpha}. (C) Levels of p53 protein were determined by western blotting. Data shown are representative of three independent experiments.

 

Figure 5
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  		Fig. 5. Effects of PFT{alpha} on BP-induced toxicity in the human leukaemic cell line KG1a. (A) KG1a cells were either untreated (UNT) or treated by 1 µM BP in the absence or presence of 20 µM PFT{alpha} for 72 h. The percentage of apoptotic Annexine V-positive cells was then evaluated as described in ‘Materials and methods’. Data are the means ± S.D. of five independent experiments. ***indicates P < 0.001, when compared with BP-treated cells not exposed to PFT{alpha}. (B) Levels of p53 protein were determined by western blotting in untreated (UNT) and BP-exposed KG1a cells; BP-treated human macrophages were used as a positive control of p53 expression. The data shown are representative of three independent experiments. (C) CYP1 mRNA levels were analysed in untreated (UNT) and BP-treated KG1a cells by RT–PCR experiments as described in ‘Materials and methods’; data shown are representative of three independent experiments. (D) TCDD-pretreated KG1a cells were exposed to 0.1 µg/ml [3H]BP for 4 h in the absence or presence of 20 µM PFT{alpha}. Adducts to cellular macromolecules due to CYP1-formed reactive BP metabolites were measured using a radiometric method as described in ‘Materials and methods’. The results are expressed as c.p.m./µg total protein and are the means ± S.D. of three independent experiments performed in duplicate. ***indicates P < 0.001, when compared with PFT{alpha}-untreated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the present study, we report that the chemical compound PFT{alpha}, isolated for its ability to suppress p53-mediated transcriptional activation (4), constitute a potent inhibitor of drug metabolizing CYP1 enzymes. Indeed, PFT{alpha} was found to inhibit CYP1-related EROD activity at concentrations already known to suppress p53 signalling (4), in both MCF-7 human mammary tumour cells and CYP1-containing liver microsomes. Using human recombinant CYP1 isoforms, PFT{alpha} was, furthermore, demonstrated to block the activity of CYP1A1, CYP1A2 and CYP1B1, with a strong inhibition of CYP1B1. Recently, among CYP1 enzymes, CYP1B1 has received considerable attention, especially owing to its wide distribution in various tissues and its recognized ability to well-activate chemical carcinogens (22). Several natural and synthetic compounds have been examined to find potent inhibitors of this CYP1 isoform (23). In this way, a marked CYP1B1 inhibition has been previously reported for the well-known CYP1 inhibitor, {alpha}-naphtoflavone (24). Analysis of kinetic parameters of CYP1B1-related EROD activity at various substrate and PFT{alpha} concentrations revealed a mixed-type inhibition (competitive and non-competitive) of human CYP1B1 by PFT{alpha}. Similarly, the food pigment purpurin has been reported to inhibit CYP1B1 activity in a mixed-type manner with a Ki value of 0.7 µM (25). Interestingly, the apparent Ki value (4.38 nM) of PFT{alpha} towards CYP1B1 is close to that of 2,4,3',5'-tetramethoxystilbene (3 nM), a methoxy derivative of resveratrol thought to be one of the most potent CYP1B1 inhibitor described until now (26), therefore, illustrating the fact that PFT{alpha} can be included among very active inhibitors of CYP1B1.

CYP1 inhibition by PFT{alpha} was not associated with a reduction of TCDD-mediated upregulation of CYP1 enzyme expression, indicating that PFT{alpha} lacks AhR antagonist properties. In fact, PFT{alpha} treatment alone rather caused an increase in CYP1 expression in MCF-7 cells, which agrees with very recent reports indicating that PFT{alpha} can induce CYP1A1 levels via activation of AhR (8,16). In contrast to PFT{alpha}, several CYP1 inhibitors such as the flavonoids, galangin (27) and resveratrol (28), block AhR activation by agonists such as TCDD and, therefore, inhibit both activity and expression of CYP1 enzymes.

As PFT{alpha} effects towards CYP1 activity were similar to those of various flavonoids, they may share some of their properties putatively contributing to their protective actions towards carcinogen activation (29). In this context, we found that inhibition of CYP1-related EROD activity by PFT{alpha} was associated with a marked reduction of CYP1-dependent conversion of BP into water-soluble metabolites and concomitantly decreased BP-induced apoptosis in human primary cultures of macrophages, shown previously to be targets for PAHs (17). Moreover, PFT{alpha} was found to completely inhibit p53 upregulation occurring in BP-treated macrophages. This was not in agreement with the established mechanism of action of PFT{alpha}, i.e. inhibition of p53 signalling without obvious alteration of p53 levels (4). Our data together with recent reports (8,16) rather indicate that PFT{alpha} protected cells from BP toxicity not through inhibiting p53 but rather through down-modulating CYP1-related formation of BP metabolites. PFT{alpha} was, furthermore, able of significantly preventing BP-derived adducts formation and BP-induced apoptosis in p53-negative human leukaemic KG1a cells, probably confirming that its protective role towards PAH toxicity was first due to down-regulation of PAH bioactivation and not to inhibition of p53 function. Interestingly, other p53-independent effects of PFT{alpha} such as inhibition of firefly luciferase activity (30), prevention of nuclear translocation of the glucocorticoid receptor (31), and activation of the AhR/AhR nuclear translocator DNA-binding complex (16) and of nuclear factor-{kappa}B transcriptional activity (32) have also been recently reported. Taken together, these data suggest that caution may be required when using PFT{alpha} as a specific p53 inhibitor and they may contribute to a re-evaluation of PFT{alpha} activity in PAH toxicity studies (6,7) or of the potential clinical use of PFT{alpha} in order to reduce side effects of chemotherapy or radiation therapy as initially proposed (5,33).

Some CYP1 inhibitors, such as resveratrol, may present an interest for chemoprevention studies through reducing bioactivation of chemical carcinogens (34). With respect to PFT{alpha}, despite its potent inhibitory effect towards CYP1B1, its anti-p53 function most likely hampered a potential use in chemoprevention owing to the critical role played by active p53 against initiation of cancerous processes (35). Development of structural analogues of PFT{alpha} retaining its CYP1B1 inhibitory effect but lacking its anti-p53 role may, however, be interesting to consider for chemoprevention studies.

In conclusion, PFT{alpha} was identified as constituting a new CYP1 inhibitor, with a potent activity towards CYP1B1. Through this action, PFT{alpha} was able to prevent toxic effects of chemical carcinogens, such as BP, requiring CYP1-related bioactivation.


   Acknowledgments
 
This work was supported by a grant from the French Agency for Sanitary and Environmental Security (AFSSE). We thank Dr D. Gilot and Dr M.A. Esnault for helpful suggestions and comments.

Conflict of Interest Statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Zedeck,M.S. (1980) Polycyclic aromatic hydrocarbons: a review. J. Environ. Pathol. Toxicol., 3, 537–567.[ISI][Medline]
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Received September 7, 2005; revised October 24, 2005; accepted October 26, 2005.


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