Deregulation of Cell Proliferation by Polycyclic Aromatic Hydrocarbons in Human Breast Carcinoma MCF-7 Cells Reflects Both Genotoxic and Non
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《毒物学科学杂志》
Department of Chemistry and Toxicology, Veterinary Research Institute, 621 32 Brno, Czech Republic; Laboratory of Cytokinetics, Institute of Biophysics, 612 65 Brno, Czech Republic; and Laboratory of Tumor Biology, Masaryk Memorial Cancer Institute, 656 53 Brno, Czech Republic
ABSTRACT
Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are carcinogens suggested to be involved in development of human cancer. Several recent studies have reported that PAHs can activate estrogen receptors (ER), either directly or indirectly by producing estrogenic metabolites. We hypothesized that the activation of ER by PAHs or their metabolites could induce cell proliferation in estrogen-sensitive cells. In the present study, we found that two PAHs, benz[a]anthracene (BaA) and BaP, can stimulate proliferation of human breast carcinoma MCF-7 cells at concentrations 100 nM and higher. This effect was ER-dependent, because it was blocked by the pure antiestrogen ICI 182,780. Although both PAHs partially inhibited S-phase entry and DNA synthesis induced by 17-estradiol, they stimulated S-phase entry when applied to MCF-7 cells synchronized by serum deprivation. This was in contrast with model antiestrogenic aryl hydrocarbon receptor ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, which fully suppressed S-phase entry. BaP, which is a strong mutagen, was found to induce p53 tumor suppressor expression, a partial S-phase arrest and at higher concentrations also cell death. Pifithrin-, a synthetic inhibitor of p53 activity, abolished both S-phase arrest and apoptosis induced by genotoxic PAHs, and it potentiated the proliferative effect of BaP. Thus, both genotoxic and nongenotoxic events seem to interact in the effects of BaP on cell proliferation. Taken together, our data indicate that both BaA and BaP can stimulate cell proliferation through activation of ER. The proliferative effects of these carcinogenic compounds might contribute to tumor promotion in estrogen-sensitive tissues.
Key Words: PAHs; estrogen receptor; p53; cell cycle; cell proliferation.
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) are a large group of diverse environmental organic pollutants formed mainly by incomplete combustion. Many of them are known or suspected carcinogens that have been reported to possess tumor-initiating and/or tumor-promoting properties (WHO, 1998). PAHs and their metabolites have been extensively studied as genotoxic, initiating agents. In contrast, their nongenotoxic or tumor promoting effects that might be linked both to carcinogenesis and endocrine disruption, are less well characterized. PAHs have been suggested to be involved in breast carcinogenesis (Burdick et al., 2003); however, the potential impact of PAHs on estrogenic signaling remains unclear. They have been reported to possess both estrogenic and antiestrogenic properties in various experimental settings. Today, PAHs are regarded mostly as antiestrogens, principally due to their ability to activate aryl hydrocarbon receptor (AhR) receptor (Arcaro et al., 1999; Chaloupka et al., 1992), which may lead to suppression of estrogen response element (ERE)-controlled gene expression via AhR-estrogen receptor- (ER) crosstalk. This could involve several mechanisms, such as a direct interaction of AhR with estrogen responsive genes, induction of inhibitory factors, competition for common nuclear coregulatory proteins or proteasome-mediated ER degradation (reviewed in Safe and Wrmke, 2003). Nevertheless, several studies have documented that some PAHs, including benzo[a]pyrene (BaP) and benz[a]anthracene (BaA) or their hydroxylated metabolites, behave like estrogenic compounds in various ER-regulated reporter gene assays (Charles et al., 2000; Clemons et al., 1998; Fertuck et al., 2001; Vondráek et al., 2002). Contrary to that, another recent study has suggested that only hydroxylated metabolites of PAHs activate transcription of endogenous estrogen-responsive genes (Gozgit et al., 2004).
Because most of the PAHs are metabolized by cytochrome P450s (CYPs) and other enzymes, large amounts of hydroxylated PAHs and quinones can form in cells (Bolton et al., 2000). Both CYP1A1 and CYP1B1 enzymes have been shown to catalyze formation of hydroxylated and dihydrodiol metabolites of PAHs, which can directly affect cell signaling through ER activation, and to serve as precursors of further active compounds, such as quinones and dihydrodiol epoxides (Shimada and Fujii-Kuriyama, 2004). The hydroxylated PAHs can activate both ER-dependent reporter genes and ER-regulated endogenous genes (Charles et al., 2000; Fertuck et al., 2001; Gozgit et al., 2004). Therefore, it might be hypothesized that they exert a stimulatory effect on proliferation of estrogen-sensitive cells and/or tissues, which is a typical hallmark of estrogenic activity (Foster et al., 2001; Soto et al., 1995). The metabolization of PAHs might also reduce the amount of parental compounds that activate AhR (Jones et al., 2000; Machala et al., 2001), which would prevent the AhR-mediated antiestrogenic effects on cell proliferation (Safe and Wrmke, 2003).
There is currently only limited information on proliferative effects of PAHs in vitro in cellular models that are being used for detection of estrogenic effects of xenobiotics, such as human breast carcinoma cell lines (Soto et al., 1995). Some PAHs, such as benzo[k]fluoranthene known as relatively potent AhR agonists, have been previously reported to act in vitro as antiestrogens in the model using postconfluent fourteen-day foci formation as an endpoint (Arcaro et al., 1999). Contrary to that, we have observed that BaA can stimulate G1-S-phase transition in serum-starved human breast carcinoma cells expressing endogenous ER (Vondráek et al., 2002). Nevertheless, the proliferation of breast epithelial cells could also be stimulated by alternative mechanisms, including, e.g., activation of epidermal growth factor receptor (EGFR), which can be activated by BaP quinones through the generation of reactive oxygen species (Burdick et al., 2003).
However, cell proliferation is an integral process reflecting multiple mechanisms activated by PAHs. The genotoxic effects of potent mutagens, such as BaP, might interfere with signaling pathways initiated by activation of either growth factor receptors or ER. BaP, which is known to form metabolites that may alter both ER and EGFR signaling, produces the ultimate carcinogenic metabolite 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (Jeffy et al., 2002; Khan and Dipple, 2000; Melendez-Colon et al., 2000). The formation of DNA adducts by genotoxic compounds may lead to activation of DNA damage checkpoints associated with G1 cell cycle arrest and activation of DNA repair mechanisms, or to induction of apoptosis in case of high level of DNA damage (Zhou and Elledge, 2000). The accumulation and transcriptional activation of p53 tumor supressor has been suggested to play an important role in cell responses to DNA damage, including cell cycle arrest, DNA repair, and apoptosis (Vousden, 2000). The exposure of human breast carcinoma cells to BaP or BPDE is known to lead to accumulation of cells in S-phase of the cell cycle (Jeffy et al., 1999; Khan and Dipple, 2000). It has been proposed that the ultimate carcinogenic metabolites of PAHs fail to induce G1 arrest and that the cells containing DNA adducts can evade G1 DNA-damage checkpoints and progress into S-phase, which increases a likelihood of malignant changes (Dipple et al., 1999; Khan and Dipple, 2000). We hypothesized that both ER activation and genotoxic events are likely to occur after exposure of MCF-7 cells to PAHs, and that the balance between genotoxic and proliferative effects of PAHs might determine the fate of cells.
Taken together, the present study aimed to investigate effects of model PAHs, with various impact on ER and/or p53 activation, on cell cycle progression and cell proliferation in ER-positive human breast carcinoma MCF-7 cells. Using inhibitors of selected signaling molecules, we attempted to describe also the mechanisms that might be involved in the effects of PAHs on cell proliferation.
MATERIALS AND METHODS
Chemicals. 17-estradiol (E2) was obtained from Sigma-Aldrich (Prague, Czech Republic). Benz[a]anthracene (BaA; CAS No. 86-73-7, purity 99.9%), benzo[a]pyrene (BaP; CAS No. 50-32-8, purity 99.9%), and fluoranthene (Fla; CAS No. 206-44-0, purity 99.9%) were purchased from Ehrenstorfer (Augsburg, Germany). Dibenzo[a,l]pyrene (DBalP; CAS No. 191-30-0, purity 99.8%) was supplied by Promochem (Wesel, Germany). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was from Cambridge Isotope Laboratories (Andover, MA). Stock solutions were prepared in dimethyl sulfoxide (DMSO) and stored at 4°C in the dark. The estrogen receptor antagonist ICI 182,780 was from Tocris (Bristol, UK). Pifithrin- was supplied by Calbiochem (San Diego, CA), and AG1478 was obtained from Sigma-Aldrich. The final concentrations of solvent in the experimental medium did not exceed 0.2% (v/v).
E-Screen assay. Human breast carcinoma MCF-7 cells, kindly provided by A. M. Soto and C. Sonnenschein (Tufts University, Boston, MA), were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich) supplemented with phenol red, NaHCO3, 10 mM HEPES, and 5% heat-inactivated fetal bovine serum (cultivation medium). The cells were grown at 37°C in the atmosphere of 5% CO2/95% air, under saturating humidity.
The MCF-7 cell proliferation assay (E-Screen) was performed according to the procedure of Soto et al. (1995) with minor modifications. MCF-7 cells retain the capacity to proliferate when estrogens are added to estrogen-free, phenol red-free DMEM, containing 5% of charcoal/dextran-treated serum (experimental medium) (Soto et al., 1995). Assays were performed in 96-well cell culture plates with initial seeding density of 3 x 103 cells per well in 100 μl of experimental medium. The cells were allowed to attach for 24 h, and 100 μl of experimental medium containing various dilutions of the tested compounds or E2 standard (positive control) were then added. We selected four model compounds that have been previously shown to exert different effects on activation of ER-dependent reporter constructs, p53 accumulation and the cell cycle: (1) BaA, a very weak mutagen which has been previously shown to stimulate the activity of ER-dependent reporter constructs (Clemons et al., 1998; Fertuck et al., 2001; Vondrácek et al., 2002); (2) BaP, which various metabolites could have both proliferative and anti-proliferative effects, and which has been reported both to activate ER-dependent reporter construct and to induce p53 accumulation (Burdick et al., 2003; Charles et al., 2000; Jeffy et al., 1999; Khan and Dipple, 2000; Vondráek et al., 2002); (3) Fla, a compound with a weak capacity to activate ER-dependent reporter construct (Vondráek et al., 2002); and (4) DBalP, a strong mutagen and carcinogen, known to induce DNA damage resulting from formation of covalent DB[a,l]PDE-DNA adducts (Melendez-Colon et al., 1999), which we have previously found to have no effect on ER-dependent reporter construct activation in MCF-7 cells (data not shown). In experiments with inhibitors, these were in all cases added simultaneously with the tested compounds. Five days later, the assay was terminated during the late exponential phase of proliferation and cell numbers were assessed using WST-1 colorimetric assay for the quantification of cell proliferation and cell viability (Roche Applied Science, Mannheim, Germany). For the WST-1 assay, 10 μl of cell proliferation reagent WST-1 were added into each well. Cells were incubated with WST-1 in cultivation conditions for another 4 h. After 4 h incubation, the spectrophotometrical absorbance of the WST-1 product was measured with a microplate spectrophotometer. To confirm the results of WST-1 assay, cell numbers were also directly determined in a selected subset of experiments. Cells were plated at a density of 8 x 104 cells per well in 6-well cell culture plates. After 24 h, MCF-7 cells were treated with DMSO, 100 pM E2, 1 μM BaA, 1 μM BaP, 1 μM Fla, or 10 nM DBalP. Five days later, cells were trypsinized and counted with a hemocytometer.
The neutral red cytotoxicity assay. The cytotoxic effects of PAHs were assessed by the neutral red cytotoxicity assay. After 24 h exposure, 100 μl of neutral red-containing medium were added into each well and plates were incubated for additional 3 h. Cells were fixated with a solution of 0.5% formaldehyde: 1% calcium chloride and the dye was extracted from the viable cells with a solution of 1% acetic acid and 50% ethanol. The plates were left to stand at room temperature for 15 min and agitated on a microplate shaker for additional 15 min. The absorbance of solubilized dye was measured at 540 nm. No acute cytotoxicity of PAHs was observed at concentrations up to 20 μM (with the exception of DBalP—the lowest experimental concentration significantly affecting cell viability was 100 nM).
Cell cycle and BrdU incorporation analyses. For serum deprivation experiments, we used 1:1 mixture of phenol red-free DMEM and Ham's F12 Nutrient Mixture (Sigma-Aldrich). For details on experimental design see legends to figures. Fixed cells were washed once with PBS and resuspended in 0.5 ml of Vindelov solution (1 M Tris-HCl – pH 8.0; 0.1% Triton X-100, v/v; 10 mM NaCl; propidium iodide 50 μg/ml; RNAse A 50 Kunitz units/ml) (Vindelov, 1977) and incubated at 37°C for 30 min. Cells were then analyzed on FACSCalibur, using 488-nm (15 mW) air cooled argon-ion laser for propidium iodide excitation, and CELLQuest software for data acquisition (Becton Dickinson, San Jose, CA). A minimum of 15,000 events was collected per sample. Data were analyzed using ModFit LT version 2.0 software (Verity Software House, Topsham, ME).
In BrdU/DNA double staining experiments, BrdU was added to cells 3 h prior to harvesting. Cells were then fixed in ethanol as described above. Fixed cells were washed once with PBS, the supernatant was removed and 1 ml HCl (2 M) with 0.5% Triton X-100 per sample was added to the pellet during continuous vortexing. Following the incubation (1 h, 37°C), samples were centrifuged, the supernatant removed, cells resuspended in 1 ml NaBO2 (pH 8.5), incubated for 10 min at room temperature, centrifuged and resuspended in 100 μl of 1% bovine serum albumin (BSA) and 0.5% Tween 20 in PBS. Cells were then incubated with FITC-conjugated mouse anti-BrdU monoclonal antibody or isotype control FITC-labeled antibody for a minimum of 1 h at 4°C in the dark. Following the incubation, 500 μl of BSA-Tween solution was added, cells were centrifuged and resuspended in 800 μl of PI solution (20 μg PI + 0.1 mg RNAse per 1 ml PBS). Cells were then incubated for 30 min at 37°C and analyzed on FACSCalibur. The excitation wavelength was 488 nm for both FITC and PI, and detection was performed with 530 nm (FITC) and 585 nm (PI) band pass filters. For quantification of BrdU positive cells, multiple events were excluded from the analyses, and the BrdU-positive region was set separately for each individual compound, based on the results of the control isotype antibody staining.
Cell death detection. Cell death induced by the tested compounds was determined using morphological criteria (fragmentation of nuclei). Cells were incubated with PAHs or vehicle as described above. In additional experiments, pifithrin- was used as a potential inhibitor of p53-mediated apoptosis (Komarov et al., 1999). After indicated time period, cells were harvested (including the floating cells) and prepared for DNA labeling with DAPI. For DAPI staining, 5 x 105 cells were resuspended with 50 μl of methanol containing 1 μg/ml DAPI (final concentration) and incubated for 30 min at room temperature. After incubation, the cells were centrifuged and mixed with 15 μl of MOWIOL (10% MOWIOL 4–88 was prepared in 25% glycerol, 100 mM Tris-HCl, pH 8.5) solution and mounted for observation under a fluorescence microscope.
Western blotting. Cells were grown in 60 mm diameter culture dishes (for 24 or 48 h) and incubated with the selected PAHs, or DMSO as the solvent control. Total cellular protein lysates were prepared by harvesting cells in lysis buffer (1% SDS, TRIS, 10% glycerol, protease inhibitor cocktail), and protein concentrations were determined with DC Protein Assay (BioRad, Hercules, CA). Proteins were then separated by SDS-polyacrylamide gel electrophoresis on 10% gel and transferred onto a nitrocellulose membrane in Bio-Rad Trans-Blot SD Semi-dry Transfer Cell (Bio-Rad Laboratories, Hercules, CA) for 75 min at room temperature applying 160 mA in transfer buffer (240 mM Tris, 190 mM glycine and 20% methanol). Prestained molecular weight markers (Bio-Rad) were run in parallel. The blotted membranes were blocked in 5% milk and 0.1% Tween 20 in PBS for 2 h at room temperature and probed overnight at 4°C with 1 μg/ml of murine monoclonal antibody DO-1 (recognizing human p53 protein; Vojtek et al., 1992) diluted in 5% milk and 0.1% Tween 20 in TBS. After washing three times in TBS plus 0.1% Tween 20, peroxidase-conjugated rabbit anti-mouse immunoglobulin antiserum (Dako, Glostrup, Denmark) diluted 1:1000 was used as the secondary antibody. To visualize peroxidase activity, ECLPlus reagents (Ammersham Pharmacia Biotech, Little Chalfont, U.K.) were used according to the manufacturer's instructions. Expression of -actin was detected with murine monoclonal antibody (clone AC-15, Sigma-Aldrich), in order to verify equal loading.
Statistical analysis. Data were expressed as means ± SD and analyzed by Student t-test, or by the nonparametric Mann-Whitney U test and Kruskal-Wallis analysis of variance (ANOVA). A p value of less than 0.05 was considered to be significant.
RESULTS
PAHs Stimulate Proliferation of MCF-7 Cells Grown in Estrogen-Free Medium
As shown in Figure 1, BaP, BaA, and Fla were all found to stimulate proliferation of MCF-7 cells grown in estrogen-free medium in a concentration-dependent manner. BaA was the most efficient inducer of proliferation of MCF-7 cells, which also potentiated the proliferation induced by E2. Contrary to that, BaP only stimulated cell proliferation at concentrations up to 1 μM, and higher concentrations of BaP decreased E2-induced cell proliferation. Fla was found to be only a weak inducer of cell proliferation, with maximum effect observed at 1 μM level (50% increase in cell numbers). DBalP decreased cell numbers both in untreated and E2-treated cells, and it was found to be cytotoxic already after 24 h at concentrations 100 nM and higher (data not shown). In order to confirm that the increased metabolic activity determined by the WST-1 assay corresponds with increased cell numbers, we also counted cells after treatment with selected PAH concentrations that induced cell proliferation. We found that all three compounds increased cell numbers when used at concentration 1 μM, and the results corresponded to those obtained in the WST-1 assay (Fig. 2).
Induction of Cell Proliferation by PAHs Is Inhibited by ICI 182,780
To specifically address the role of ER and EGFR in the observed stimulation of cell proliferation, we employed synthetic antiestrogen ICI 182,780 (Wakeling, 1995) and AG1478, which is a specific inhibitor of EGFR kinase that has been shown to block the EGFR-mediated cell proliferation in human breast epithelial MCF-10A cells (Burdick et al., 2003; Levitzki and Gazit, 1995). As shown in Figure 3A, ICI 182,780 supressed cell proliferation induced by E2 and all three PAHs tested—BaA, BaP, and Fla. Contrary to that, AG1478 had no effect either on E2-induced or on PAH-induced stimulation of cell proliferation (Fig. 3B). We also tested, a specific inhibitor of activation of MEK1/2 known to participate in EGFR signaling, which had no significant effect on cell proliferation (data not shown). These data suggested that ER was responsible for induction of cell proliferation by PAHs in MCF-7 cells.
BaP and DBalP, but Not BaA, Induce p53 Accumulation, S-Phase Arrest, and Cell Death in MCF-7 Cells
We hypothesized that the balance between genotoxic and proliferative effects of various PAH metabolites might determine the overall impact of PAHs on cell proliferation. BaA is known to be a relatively weak mutagen as compared to both BaP and DBalP. Because it has been suggested that induction of p53 expression reflects the formation of DNA adducts in exposed cells (Ramet et al., 1995), we examined effects of BaA, BaP, and DBalP on p53 expression in cells grown either in estrogen-free environment or incubated with 100 pM E2. As shown in Figure 4A, both BaP and DBalP, but not BaA, induced p53 accumulation in MCF-7 cells. These effects were similar both in the E2-treated cells and in the cells treated only with BaP or DBalP. As p53 accumulation has been reported to be associated with PAH induction of cell cycle arrest in S-phase and/or induction of apoptosis in various cell lines, we next investigated effects of all three PAHs on cell cycle and cell death following the 72-h incubation with tested compounds. The results are outlined in Figures 4B and 4C. Both BaP and DBalP induced a significant accumulation of cells in S-phase. In contrast, BaA only induced a marginal increase of percentage of cells in S-phase after treatment with 10 μM concentration in MCF-7 cells, which might be associated with its proliferative activity. Fragmented nuclei were detected after DBalP treatment, while BaP induced cell death only at concentration 10 μM, and BaA did not induce apoptosis.
In order to discriminate between genotoxic and proliferative effects induced by BaP, we chose to use pifithrin-, inhibitor of genotoxic events associated with p53 activation, which has been successfully used to prevent apoptosis induced by both BaP and DBalP (Chramostová et al., 2004; Komarov et al., 1999). As shown in Figures 4B and 4C, pifithrin- prevented S-phase arrest induced by BaP and DBalP, as well as the induction of cell death by DBalP. Pifithrin- had no effect on cell proliferation in control, E2- or BaA-treated cells, while it significantly potentiated survival of DBalP-treated cells and it increased numbers of BaP-treated cells (Fig. 5). These results suggested that BaP induced both genotoxic and nongenotoxic effects in MCF-7 cells, and that the latter could prevail when genotoxic events were inhibited.
Modulation of Cell Cycle Progression and DNA Synthesis by PAHs and High Efficiency AhR Ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
Given that the genotoxic PAHs were observed to inhibit S-phase progression in MCF-7 cells, we were interested in determining the effects of PAHs on cell cycle progression of MCF-7 cells synchronized by serum deprivation. We found that BaP, DBalP, and BaA induced a dose-dependent increase of percentage of cells in S-phase following the 24-h treatment (Fig. 6). Because we wanted to discriminate between induction of G1-S-phase transition, which could be associated with ER activation, and delay in S-phase induced by genotoxic events, we next investigated distribution of cells in S-phase using dual anti-BrdU/PI staining. The results are shown in Figure 7. We observed an increase of percentage of BrdU-positive S-phase cells among the cells treated with PAHs (BaP 1 μM, BaA 1 μM, DBalP 10 nM). However, the distribution between early and late S-phase of BrdU-positive cells was different among the tested compounds. BaP and DBalP, but not BaA, induced a delay in S-phase progression, as evidenced by an increased percentage of cells found in early S-phase region. This trend was apparent both in the E2-stimulated cells and in the cells treated with PAHs alone. This confirmed that BaA did not activate genotoxic mechanisms associated with formation of DNA-adducts and p53 accumulation in MCF-7 cells, which may lead to S-phase arrest.
Strong AhR ligands, such as TCDD, can reportedly inhibit G1-S-phase progression and block cell proliferation (Wang et al., 1998), yet we found that both BaP and BaA can stimulate cell proliferation in MCF-7 cells. Therefore, we compared effects of BaP, BaA, and TCDD on cell cycle progression. As shown in Figure 7, 5 nM TCDD decreased the percentage of BrdU-positive cells, and it inhibited the E2-stimulated cell cycle progression. Both BaA and BaP also induced a decrease of total percentage of S-phase of E2-treated cells, when compared with the group treated with E2 alone. However, unlike TCDD, both BaA and BaP stimulated G1-S-phase transition in cells treated with PAHs alone. This seems to suggest that both compounds exerted a dual type of effect on MCF-7 cells—while partially inhibiting cell cycle progression in cells treated with E2 as a strong mitogen, both compounds stimulated S-phase entry in cells synchronized by serum deprivation.
DISCUSSION
Perturbation of the balance between cell proliferation and apoptosis have been suggested to play a significant role in mediating and modifying the action of carcinogens in various target organs (Roberts et al., 1997). The increased proliferative rate may increase probability of initiated clonal cell expansion and increase the risk of DNA modification during its replication (Dipple et al., 1999; Klaunig et al., 2000). It has been hypothesized that PAHs, being both widespread environmental pollutants and important cigarette smoke constituents, could be involved in breast carcinogenesis as one of environmental risk factors (Burdick et al., 2003; Hecht, 2002). However, the available data on induction of cell proliferation by PAHs in breast epithelial cells are still not sufficient and often controversial, although this mode of action could contribute to tumor promotion. PAHs, or their hydroxylated metabolites, can act as estrogens in various estrogenicity assays (Charles et al., 2000; Clemons et al., 1998; Fertuck et al., 2001; Gozgit et al., 2004; Vondráek et al., 2002). Nevertheless, it has been suggested that only hydroxylated forms of PAHs are able to induce a transcription of endogenous ER-regulated genes (Gozgit et al., 2004), and some PAHs have been proposed to act as antiestrogens (Arcaro et al., 1999; Chaloupka et al., 1992), in a manner similar to other AhR ligands (Safe and Wrmke, 2003; Wang et al., 1998). Contrary to that, BaA can stimulate cell cycle progression in human breast carcinoma cells in an ER-dependent manner (Vondráek et al., 2002), and BaP quinones can activate EGFR in the ER-negative MCF-10A human breast epithelial cell line, which may also lead to induction of cell proliferation (Burdick et al., 2003). In the present study, we investigated effects of selected PAHs that have been previously shown to either directly or indirectly (by producing estrogenic metabolites) activate ER, on proliferation of human breast carcinoma MCF-7 cells, which express high levels of endogenous ER.
The major finding of this study was that both BaA and BaP induced proliferation of MCF-7 cells grown in estrogen-free medium, at concentrations as low as 100 nM. The third compound tested, Fla, was only a weak inducer of cell proliferation, increasing cell numbers by approximately 50% at 1 μM concentration (Figs. 1 and 2). Both BaA and BaP are carcinogenic compounds, listed as suspected human carcinogens (WHO, 1998). However, unlike BaP, BaA is a relatively weak mutagen, which produces a significantly lower amount of DNA-adducts than BaP in human cells (Binková and rám, 2004). Our data suggest that induction of cell proliferation by BaA was mediated by ER, because it was prevented by antiestrogen ICI 182,780. BaA was also found to increase DNA-synthesis and to promote G1-S-phase cell cycle transition in serum-deprived MCF-7 cells. Contrary to that, BaA did not induce p53 expression, confirming that it is only a weak genotoxin in MCF-7 cells. Taken together, the experimental evidence presented in this study seem to suggest that BaA can stimulate cell proliferation in estrogen-sensitive cells. This mechanism could contribute to its carcinogenic effects.
In contrast to BaA, BaP is a potent mutagen which is known to produce large amounts of BPDE-DNA adducts, which in turn leads to S-phase arrest/delay and induction of apoptosis (Binková and Sram, 2004; Chen et al., 2003; Jeffy et al., 1999; Khan and Dipple, 2000). BaP has been also suggested to produce quinone metabolites leading to induction of oxidative stress, associated both with oxidative DNA damage and transactivation of EGFR (Bolton et al., 2000; Burdick et al., 2003). In the present study, BaP induced S-phase arrest and at high concentrations also cell death, which is in accordance with the above studies. However, we found that BaP also stimulated proliferation of MCF-7 cells within concentration range 100 nM–1 μM. This proliferative effect was abolished by ICI 182,780. This is in accordance with a recent study showing that BaP can induce ER-dependent cell prolieration in vitro (Tsai et. al., 2004). Pifithrin-, which prevented induction of S-phase arrest, was found to potentiate cell proliferation induced by BaP (Fig. 5). It did not increase cell numbers in DBalP-treated group, suggesting that DBalP (a model genotoxic compound), in contrast to BaP, does not activate ER-driven processes in MCF-7 cells. A specific inhibitor of EGFR kinase had no effect either on BaP- or on BaA-stimulated cell proliferation, and EGFR was probably not involved in the observed stimulation of cell proliferation. Thus, our data showed that BaP can stimulate cell proliferation in MCF-7 cells by a mechanism involving ER activation, which is further potentiated by p53 inhibitor. Induction of cell proliferation and DNA synthesis could further contribute to the proposed "stealth properties" of BaP as a carcinogen, and to increase a likelihood of DNA replication on a damaged template (Dipple et al., 1999). Nevertheless, the present data also show that the genotoxic events leading to cell cycle arrest and/or cell death would decrease cell numbers, thus leading to overestimation of antiestrogenicity of mutagenic compounds, such as PAHs.
The antiestrogenicity of PAHs has been suggested to be associated with the activation of AhR (Chaloupka et al., 1992; Gozgit et al., 2004; Safe and Wrmke, 2003). Both BaA and BaP have been reported to inhibit foci development in MCF-7 cell cultures (Arcaro et al., 1999). This seems to be in contrast with our data, however, it should be mentioned that conditions used for cell growth in the present study were significantly different from the postconfluent cell model, and different experimental settings might be responsible for these discrepancies. Because AhR ligands, such as TCDD, are known to exert their anti-proliferative effects at least in part via inhibition of G1-S-phase transition (Wang et al., 1998), we compared the effects of model PAHs and TCDD on cell cycle progression in MCF-7 cells synchronized by serum deprivation. Using a combination of DNA staining and detection of BrdU incorporation, we found that like TCDD, BaP and BaA also partially inhibited induction of S-phase entry by E2. However, unlike TCDD, both BaP and BaA also stimulated G1-S-phase transition, when applied to serum-starved cells, albeit to a lesser extent than E2 itself. Interestingly, dibenzo[a,h]anthracene, a strong AhR ligand, which has been shown to be antiestrogenic in MCF-7 cells (Arcaro et al., 1999), had the same effect as TCDD both on the E2-treated and untreated cells (data not shown). These results seem to support the hypothesis that unlike other PAHs, both BaP and BaA, or their metabolites that are less efficient inducers of AhR-mediated activity, can activate ER and stimulate cell proliferation. Recently, 6-methyl-1,3,8-trichlorodibenzofuran, a weak agonist and a partial antagonist of AhR, which is currently being tested for its potential therapeutic use, has been found to activate ER and to stimulate MCF-7 cells proliferation, suggesting that this dual type of activity can be observed with some compounds (Pearce et al., 2004).
High levels of both BaP and BaA can be found in the respirable particulate matter of atmospheric pollution in urban environment. Both compounds were determined to be present at concentrations of several hundreds of ng per mg of extractable organic matter in urban air samples (Binková et al., 2003; Gábelová et al., 2004). Moreover, both compounds are important cigarette smoke constituents (Hoffmann et al., 2001), and they represent a significant part of carcinogenic PAHs found in environmental samples (Gábelová et al., 2004). Therefore, they might be considered to pose a potential threat to human health. Due to the rapid metabolization of PAHs, there is currently not sufficient information on levels of PAHs or their metabolites in tissues, such as human mammary tissue. Although there is presently no evidence that specific tobacco smoke carcinogens reach human mammary tissue, there is convincing evidence that mammary carcinogens in tobacco smoke are taken up and metabolized in smokers (Hecht, 2002). It should be noted, however, that exposure to BaP and BaA occurs simultaneously with exposure to high amounts of other PAHs, including both mutagens and efficient AhR ligands. This suggests that the proliferative effects of BaP and BaA could be modulated also by antiestrogenic or genotoxic effects of other pollutants present in complex environmental mixtures or in cigarette smoke.
In conclusion, we found that two PAHs, BaA and BaP, can stimulate proliferation of human breast carcinoma MCF-7 cells in ER-dependent manner. In contrast to TCDD, a model persistent AhR ligand, both BaP and BaA stimulated S-phase entry when applied to G1-synchronized MCF-7 cells. These results seem to suggest that inhibitory effects of BaP and BaA on cell proliferation as AhR ligands could be overcome by activation of ER. Unlike BaA, BaP induced p53 expression, a partial S-phase arrest and cell death at high concentrations. Pifithrin- abolished both S-phase arrest and cell death, and it potentiated the proliferative effect of BaP, suggesting that both genotoxic and nongenotoxic events may interact in effects of BaP on cell proliferation. The proliferative effects of both carcinogenic compounds might contribute to tumor promotion and, ultimately, carcinogenesis in estrogen-sensitive tissues. However, whether this phenomenon is restricted to the particular cell line studied here, or whether it is of in vivo relevance remains to be elucidated.
ACKNOWLEDGMENTS
Authors thank Prof. Ana M. Soto and Prof. Carlos Sonnenschein (Tufts University, Boston, MA) for providing MCF-7 cell line. This work was supported by the Czech Science Foundation (grants no. 525/01/D076 and 525/03/1527) and by the Research Plan of the Academy of Sciences of the Czech Republic No. Z5004920.
NOTES
1 These authors contributed equally to this work.
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ABSTRACT
Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are carcinogens suggested to be involved in development of human cancer. Several recent studies have reported that PAHs can activate estrogen receptors (ER), either directly or indirectly by producing estrogenic metabolites. We hypothesized that the activation of ER by PAHs or their metabolites could induce cell proliferation in estrogen-sensitive cells. In the present study, we found that two PAHs, benz[a]anthracene (BaA) and BaP, can stimulate proliferation of human breast carcinoma MCF-7 cells at concentrations 100 nM and higher. This effect was ER-dependent, because it was blocked by the pure antiestrogen ICI 182,780. Although both PAHs partially inhibited S-phase entry and DNA synthesis induced by 17-estradiol, they stimulated S-phase entry when applied to MCF-7 cells synchronized by serum deprivation. This was in contrast with model antiestrogenic aryl hydrocarbon receptor ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, which fully suppressed S-phase entry. BaP, which is a strong mutagen, was found to induce p53 tumor suppressor expression, a partial S-phase arrest and at higher concentrations also cell death. Pifithrin-, a synthetic inhibitor of p53 activity, abolished both S-phase arrest and apoptosis induced by genotoxic PAHs, and it potentiated the proliferative effect of BaP. Thus, both genotoxic and nongenotoxic events seem to interact in the effects of BaP on cell proliferation. Taken together, our data indicate that both BaA and BaP can stimulate cell proliferation through activation of ER. The proliferative effects of these carcinogenic compounds might contribute to tumor promotion in estrogen-sensitive tissues.
Key Words: PAHs; estrogen receptor; p53; cell cycle; cell proliferation.
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs) are a large group of diverse environmental organic pollutants formed mainly by incomplete combustion. Many of them are known or suspected carcinogens that have been reported to possess tumor-initiating and/or tumor-promoting properties (WHO, 1998). PAHs and their metabolites have been extensively studied as genotoxic, initiating agents. In contrast, their nongenotoxic or tumor promoting effects that might be linked both to carcinogenesis and endocrine disruption, are less well characterized. PAHs have been suggested to be involved in breast carcinogenesis (Burdick et al., 2003); however, the potential impact of PAHs on estrogenic signaling remains unclear. They have been reported to possess both estrogenic and antiestrogenic properties in various experimental settings. Today, PAHs are regarded mostly as antiestrogens, principally due to their ability to activate aryl hydrocarbon receptor (AhR) receptor (Arcaro et al., 1999; Chaloupka et al., 1992), which may lead to suppression of estrogen response element (ERE)-controlled gene expression via AhR-estrogen receptor- (ER) crosstalk. This could involve several mechanisms, such as a direct interaction of AhR with estrogen responsive genes, induction of inhibitory factors, competition for common nuclear coregulatory proteins or proteasome-mediated ER degradation (reviewed in Safe and Wrmke, 2003). Nevertheless, several studies have documented that some PAHs, including benzo[a]pyrene (BaP) and benz[a]anthracene (BaA) or their hydroxylated metabolites, behave like estrogenic compounds in various ER-regulated reporter gene assays (Charles et al., 2000; Clemons et al., 1998; Fertuck et al., 2001; Vondráek et al., 2002). Contrary to that, another recent study has suggested that only hydroxylated metabolites of PAHs activate transcription of endogenous estrogen-responsive genes (Gozgit et al., 2004).
Because most of the PAHs are metabolized by cytochrome P450s (CYPs) and other enzymes, large amounts of hydroxylated PAHs and quinones can form in cells (Bolton et al., 2000). Both CYP1A1 and CYP1B1 enzymes have been shown to catalyze formation of hydroxylated and dihydrodiol metabolites of PAHs, which can directly affect cell signaling through ER activation, and to serve as precursors of further active compounds, such as quinones and dihydrodiol epoxides (Shimada and Fujii-Kuriyama, 2004). The hydroxylated PAHs can activate both ER-dependent reporter genes and ER-regulated endogenous genes (Charles et al., 2000; Fertuck et al., 2001; Gozgit et al., 2004). Therefore, it might be hypothesized that they exert a stimulatory effect on proliferation of estrogen-sensitive cells and/or tissues, which is a typical hallmark of estrogenic activity (Foster et al., 2001; Soto et al., 1995). The metabolization of PAHs might also reduce the amount of parental compounds that activate AhR (Jones et al., 2000; Machala et al., 2001), which would prevent the AhR-mediated antiestrogenic effects on cell proliferation (Safe and Wrmke, 2003).
There is currently only limited information on proliferative effects of PAHs in vitro in cellular models that are being used for detection of estrogenic effects of xenobiotics, such as human breast carcinoma cell lines (Soto et al., 1995). Some PAHs, such as benzo[k]fluoranthene known as relatively potent AhR agonists, have been previously reported to act in vitro as antiestrogens in the model using postconfluent fourteen-day foci formation as an endpoint (Arcaro et al., 1999). Contrary to that, we have observed that BaA can stimulate G1-S-phase transition in serum-starved human breast carcinoma cells expressing endogenous ER (Vondráek et al., 2002). Nevertheless, the proliferation of breast epithelial cells could also be stimulated by alternative mechanisms, including, e.g., activation of epidermal growth factor receptor (EGFR), which can be activated by BaP quinones through the generation of reactive oxygen species (Burdick et al., 2003).
However, cell proliferation is an integral process reflecting multiple mechanisms activated by PAHs. The genotoxic effects of potent mutagens, such as BaP, might interfere with signaling pathways initiated by activation of either growth factor receptors or ER. BaP, which is known to form metabolites that may alter both ER and EGFR signaling, produces the ultimate carcinogenic metabolite 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) (Jeffy et al., 2002; Khan and Dipple, 2000; Melendez-Colon et al., 2000). The formation of DNA adducts by genotoxic compounds may lead to activation of DNA damage checkpoints associated with G1 cell cycle arrest and activation of DNA repair mechanisms, or to induction of apoptosis in case of high level of DNA damage (Zhou and Elledge, 2000). The accumulation and transcriptional activation of p53 tumor supressor has been suggested to play an important role in cell responses to DNA damage, including cell cycle arrest, DNA repair, and apoptosis (Vousden, 2000). The exposure of human breast carcinoma cells to BaP or BPDE is known to lead to accumulation of cells in S-phase of the cell cycle (Jeffy et al., 1999; Khan and Dipple, 2000). It has been proposed that the ultimate carcinogenic metabolites of PAHs fail to induce G1 arrest and that the cells containing DNA adducts can evade G1 DNA-damage checkpoints and progress into S-phase, which increases a likelihood of malignant changes (Dipple et al., 1999; Khan and Dipple, 2000). We hypothesized that both ER activation and genotoxic events are likely to occur after exposure of MCF-7 cells to PAHs, and that the balance between genotoxic and proliferative effects of PAHs might determine the fate of cells.
Taken together, the present study aimed to investigate effects of model PAHs, with various impact on ER and/or p53 activation, on cell cycle progression and cell proliferation in ER-positive human breast carcinoma MCF-7 cells. Using inhibitors of selected signaling molecules, we attempted to describe also the mechanisms that might be involved in the effects of PAHs on cell proliferation.
MATERIALS AND METHODS
Chemicals. 17-estradiol (E2) was obtained from Sigma-Aldrich (Prague, Czech Republic). Benz[a]anthracene (BaA; CAS No. 86-73-7, purity 99.9%), benzo[a]pyrene (BaP; CAS No. 50-32-8, purity 99.9%), and fluoranthene (Fla; CAS No. 206-44-0, purity 99.9%) were purchased from Ehrenstorfer (Augsburg, Germany). Dibenzo[a,l]pyrene (DBalP; CAS No. 191-30-0, purity 99.8%) was supplied by Promochem (Wesel, Germany). 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was from Cambridge Isotope Laboratories (Andover, MA). Stock solutions were prepared in dimethyl sulfoxide (DMSO) and stored at 4°C in the dark. The estrogen receptor antagonist ICI 182,780 was from Tocris (Bristol, UK). Pifithrin- was supplied by Calbiochem (San Diego, CA), and AG1478 was obtained from Sigma-Aldrich. The final concentrations of solvent in the experimental medium did not exceed 0.2% (v/v).
E-Screen assay. Human breast carcinoma MCF-7 cells, kindly provided by A. M. Soto and C. Sonnenschein (Tufts University, Boston, MA), were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Sigma-Aldrich) supplemented with phenol red, NaHCO3, 10 mM HEPES, and 5% heat-inactivated fetal bovine serum (cultivation medium). The cells were grown at 37°C in the atmosphere of 5% CO2/95% air, under saturating humidity.
The MCF-7 cell proliferation assay (E-Screen) was performed according to the procedure of Soto et al. (1995) with minor modifications. MCF-7 cells retain the capacity to proliferate when estrogens are added to estrogen-free, phenol red-free DMEM, containing 5% of charcoal/dextran-treated serum (experimental medium) (Soto et al., 1995). Assays were performed in 96-well cell culture plates with initial seeding density of 3 x 103 cells per well in 100 μl of experimental medium. The cells were allowed to attach for 24 h, and 100 μl of experimental medium containing various dilutions of the tested compounds or E2 standard (positive control) were then added. We selected four model compounds that have been previously shown to exert different effects on activation of ER-dependent reporter constructs, p53 accumulation and the cell cycle: (1) BaA, a very weak mutagen which has been previously shown to stimulate the activity of ER-dependent reporter constructs (Clemons et al., 1998; Fertuck et al., 2001; Vondrácek et al., 2002); (2) BaP, which various metabolites could have both proliferative and anti-proliferative effects, and which has been reported both to activate ER-dependent reporter construct and to induce p53 accumulation (Burdick et al., 2003; Charles et al., 2000; Jeffy et al., 1999; Khan and Dipple, 2000; Vondráek et al., 2002); (3) Fla, a compound with a weak capacity to activate ER-dependent reporter construct (Vondráek et al., 2002); and (4) DBalP, a strong mutagen and carcinogen, known to induce DNA damage resulting from formation of covalent DB[a,l]PDE-DNA adducts (Melendez-Colon et al., 1999), which we have previously found to have no effect on ER-dependent reporter construct activation in MCF-7 cells (data not shown). In experiments with inhibitors, these were in all cases added simultaneously with the tested compounds. Five days later, the assay was terminated during the late exponential phase of proliferation and cell numbers were assessed using WST-1 colorimetric assay for the quantification of cell proliferation and cell viability (Roche Applied Science, Mannheim, Germany). For the WST-1 assay, 10 μl of cell proliferation reagent WST-1 were added into each well. Cells were incubated with WST-1 in cultivation conditions for another 4 h. After 4 h incubation, the spectrophotometrical absorbance of the WST-1 product was measured with a microplate spectrophotometer. To confirm the results of WST-1 assay, cell numbers were also directly determined in a selected subset of experiments. Cells were plated at a density of 8 x 104 cells per well in 6-well cell culture plates. After 24 h, MCF-7 cells were treated with DMSO, 100 pM E2, 1 μM BaA, 1 μM BaP, 1 μM Fla, or 10 nM DBalP. Five days later, cells were trypsinized and counted with a hemocytometer.
The neutral red cytotoxicity assay. The cytotoxic effects of PAHs were assessed by the neutral red cytotoxicity assay. After 24 h exposure, 100 μl of neutral red-containing medium were added into each well and plates were incubated for additional 3 h. Cells were fixated with a solution of 0.5% formaldehyde: 1% calcium chloride and the dye was extracted from the viable cells with a solution of 1% acetic acid and 50% ethanol. The plates were left to stand at room temperature for 15 min and agitated on a microplate shaker for additional 15 min. The absorbance of solubilized dye was measured at 540 nm. No acute cytotoxicity of PAHs was observed at concentrations up to 20 μM (with the exception of DBalP—the lowest experimental concentration significantly affecting cell viability was 100 nM).
Cell cycle and BrdU incorporation analyses. For serum deprivation experiments, we used 1:1 mixture of phenol red-free DMEM and Ham's F12 Nutrient Mixture (Sigma-Aldrich). For details on experimental design see legends to figures. Fixed cells were washed once with PBS and resuspended in 0.5 ml of Vindelov solution (1 M Tris-HCl – pH 8.0; 0.1% Triton X-100, v/v; 10 mM NaCl; propidium iodide 50 μg/ml; RNAse A 50 Kunitz units/ml) (Vindelov, 1977) and incubated at 37°C for 30 min. Cells were then analyzed on FACSCalibur, using 488-nm (15 mW) air cooled argon-ion laser for propidium iodide excitation, and CELLQuest software for data acquisition (Becton Dickinson, San Jose, CA). A minimum of 15,000 events was collected per sample. Data were analyzed using ModFit LT version 2.0 software (Verity Software House, Topsham, ME).
In BrdU/DNA double staining experiments, BrdU was added to cells 3 h prior to harvesting. Cells were then fixed in ethanol as described above. Fixed cells were washed once with PBS, the supernatant was removed and 1 ml HCl (2 M) with 0.5% Triton X-100 per sample was added to the pellet during continuous vortexing. Following the incubation (1 h, 37°C), samples were centrifuged, the supernatant removed, cells resuspended in 1 ml NaBO2 (pH 8.5), incubated for 10 min at room temperature, centrifuged and resuspended in 100 μl of 1% bovine serum albumin (BSA) and 0.5% Tween 20 in PBS. Cells were then incubated with FITC-conjugated mouse anti-BrdU monoclonal antibody or isotype control FITC-labeled antibody for a minimum of 1 h at 4°C in the dark. Following the incubation, 500 μl of BSA-Tween solution was added, cells were centrifuged and resuspended in 800 μl of PI solution (20 μg PI + 0.1 mg RNAse per 1 ml PBS). Cells were then incubated for 30 min at 37°C and analyzed on FACSCalibur. The excitation wavelength was 488 nm for both FITC and PI, and detection was performed with 530 nm (FITC) and 585 nm (PI) band pass filters. For quantification of BrdU positive cells, multiple events were excluded from the analyses, and the BrdU-positive region was set separately for each individual compound, based on the results of the control isotype antibody staining.
Cell death detection. Cell death induced by the tested compounds was determined using morphological criteria (fragmentation of nuclei). Cells were incubated with PAHs or vehicle as described above. In additional experiments, pifithrin- was used as a potential inhibitor of p53-mediated apoptosis (Komarov et al., 1999). After indicated time period, cells were harvested (including the floating cells) and prepared for DNA labeling with DAPI. For DAPI staining, 5 x 105 cells were resuspended with 50 μl of methanol containing 1 μg/ml DAPI (final concentration) and incubated for 30 min at room temperature. After incubation, the cells were centrifuged and mixed with 15 μl of MOWIOL (10% MOWIOL 4–88 was prepared in 25% glycerol, 100 mM Tris-HCl, pH 8.5) solution and mounted for observation under a fluorescence microscope.
Western blotting. Cells were grown in 60 mm diameter culture dishes (for 24 or 48 h) and incubated with the selected PAHs, or DMSO as the solvent control. Total cellular protein lysates were prepared by harvesting cells in lysis buffer (1% SDS, TRIS, 10% glycerol, protease inhibitor cocktail), and protein concentrations were determined with DC Protein Assay (BioRad, Hercules, CA). Proteins were then separated by SDS-polyacrylamide gel electrophoresis on 10% gel and transferred onto a nitrocellulose membrane in Bio-Rad Trans-Blot SD Semi-dry Transfer Cell (Bio-Rad Laboratories, Hercules, CA) for 75 min at room temperature applying 160 mA in transfer buffer (240 mM Tris, 190 mM glycine and 20% methanol). Prestained molecular weight markers (Bio-Rad) were run in parallel. The blotted membranes were blocked in 5% milk and 0.1% Tween 20 in PBS for 2 h at room temperature and probed overnight at 4°C with 1 μg/ml of murine monoclonal antibody DO-1 (recognizing human p53 protein; Vojtek et al., 1992) diluted in 5% milk and 0.1% Tween 20 in TBS. After washing three times in TBS plus 0.1% Tween 20, peroxidase-conjugated rabbit anti-mouse immunoglobulin antiserum (Dako, Glostrup, Denmark) diluted 1:1000 was used as the secondary antibody. To visualize peroxidase activity, ECLPlus reagents (Ammersham Pharmacia Biotech, Little Chalfont, U.K.) were used according to the manufacturer's instructions. Expression of -actin was detected with murine monoclonal antibody (clone AC-15, Sigma-Aldrich), in order to verify equal loading.
Statistical analysis. Data were expressed as means ± SD and analyzed by Student t-test, or by the nonparametric Mann-Whitney U test and Kruskal-Wallis analysis of variance (ANOVA). A p value of less than 0.05 was considered to be significant.
RESULTS
PAHs Stimulate Proliferation of MCF-7 Cells Grown in Estrogen-Free Medium
As shown in Figure 1, BaP, BaA, and Fla were all found to stimulate proliferation of MCF-7 cells grown in estrogen-free medium in a concentration-dependent manner. BaA was the most efficient inducer of proliferation of MCF-7 cells, which also potentiated the proliferation induced by E2. Contrary to that, BaP only stimulated cell proliferation at concentrations up to 1 μM, and higher concentrations of BaP decreased E2-induced cell proliferation. Fla was found to be only a weak inducer of cell proliferation, with maximum effect observed at 1 μM level (50% increase in cell numbers). DBalP decreased cell numbers both in untreated and E2-treated cells, and it was found to be cytotoxic already after 24 h at concentrations 100 nM and higher (data not shown). In order to confirm that the increased metabolic activity determined by the WST-1 assay corresponds with increased cell numbers, we also counted cells after treatment with selected PAH concentrations that induced cell proliferation. We found that all three compounds increased cell numbers when used at concentration 1 μM, and the results corresponded to those obtained in the WST-1 assay (Fig. 2).
Induction of Cell Proliferation by PAHs Is Inhibited by ICI 182,780
To specifically address the role of ER and EGFR in the observed stimulation of cell proliferation, we employed synthetic antiestrogen ICI 182,780 (Wakeling, 1995) and AG1478, which is a specific inhibitor of EGFR kinase that has been shown to block the EGFR-mediated cell proliferation in human breast epithelial MCF-10A cells (Burdick et al., 2003; Levitzki and Gazit, 1995). As shown in Figure 3A, ICI 182,780 supressed cell proliferation induced by E2 and all three PAHs tested—BaA, BaP, and Fla. Contrary to that, AG1478 had no effect either on E2-induced or on PAH-induced stimulation of cell proliferation (Fig. 3B). We also tested, a specific inhibitor of activation of MEK1/2 known to participate in EGFR signaling, which had no significant effect on cell proliferation (data not shown). These data suggested that ER was responsible for induction of cell proliferation by PAHs in MCF-7 cells.
BaP and DBalP, but Not BaA, Induce p53 Accumulation, S-Phase Arrest, and Cell Death in MCF-7 Cells
We hypothesized that the balance between genotoxic and proliferative effects of various PAH metabolites might determine the overall impact of PAHs on cell proliferation. BaA is known to be a relatively weak mutagen as compared to both BaP and DBalP. Because it has been suggested that induction of p53 expression reflects the formation of DNA adducts in exposed cells (Ramet et al., 1995), we examined effects of BaA, BaP, and DBalP on p53 expression in cells grown either in estrogen-free environment or incubated with 100 pM E2. As shown in Figure 4A, both BaP and DBalP, but not BaA, induced p53 accumulation in MCF-7 cells. These effects were similar both in the E2-treated cells and in the cells treated only with BaP or DBalP. As p53 accumulation has been reported to be associated with PAH induction of cell cycle arrest in S-phase and/or induction of apoptosis in various cell lines, we next investigated effects of all three PAHs on cell cycle and cell death following the 72-h incubation with tested compounds. The results are outlined in Figures 4B and 4C. Both BaP and DBalP induced a significant accumulation of cells in S-phase. In contrast, BaA only induced a marginal increase of percentage of cells in S-phase after treatment with 10 μM concentration in MCF-7 cells, which might be associated with its proliferative activity. Fragmented nuclei were detected after DBalP treatment, while BaP induced cell death only at concentration 10 μM, and BaA did not induce apoptosis.
In order to discriminate between genotoxic and proliferative effects induced by BaP, we chose to use pifithrin-, inhibitor of genotoxic events associated with p53 activation, which has been successfully used to prevent apoptosis induced by both BaP and DBalP (Chramostová et al., 2004; Komarov et al., 1999). As shown in Figures 4B and 4C, pifithrin- prevented S-phase arrest induced by BaP and DBalP, as well as the induction of cell death by DBalP. Pifithrin- had no effect on cell proliferation in control, E2- or BaA-treated cells, while it significantly potentiated survival of DBalP-treated cells and it increased numbers of BaP-treated cells (Fig. 5). These results suggested that BaP induced both genotoxic and nongenotoxic effects in MCF-7 cells, and that the latter could prevail when genotoxic events were inhibited.
Modulation of Cell Cycle Progression and DNA Synthesis by PAHs and High Efficiency AhR Ligand, 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)
Given that the genotoxic PAHs were observed to inhibit S-phase progression in MCF-7 cells, we were interested in determining the effects of PAHs on cell cycle progression of MCF-7 cells synchronized by serum deprivation. We found that BaP, DBalP, and BaA induced a dose-dependent increase of percentage of cells in S-phase following the 24-h treatment (Fig. 6). Because we wanted to discriminate between induction of G1-S-phase transition, which could be associated with ER activation, and delay in S-phase induced by genotoxic events, we next investigated distribution of cells in S-phase using dual anti-BrdU/PI staining. The results are shown in Figure 7. We observed an increase of percentage of BrdU-positive S-phase cells among the cells treated with PAHs (BaP 1 μM, BaA 1 μM, DBalP 10 nM). However, the distribution between early and late S-phase of BrdU-positive cells was different among the tested compounds. BaP and DBalP, but not BaA, induced a delay in S-phase progression, as evidenced by an increased percentage of cells found in early S-phase region. This trend was apparent both in the E2-stimulated cells and in the cells treated with PAHs alone. This confirmed that BaA did not activate genotoxic mechanisms associated with formation of DNA-adducts and p53 accumulation in MCF-7 cells, which may lead to S-phase arrest.
Strong AhR ligands, such as TCDD, can reportedly inhibit G1-S-phase progression and block cell proliferation (Wang et al., 1998), yet we found that both BaP and BaA can stimulate cell proliferation in MCF-7 cells. Therefore, we compared effects of BaP, BaA, and TCDD on cell cycle progression. As shown in Figure 7, 5 nM TCDD decreased the percentage of BrdU-positive cells, and it inhibited the E2-stimulated cell cycle progression. Both BaA and BaP also induced a decrease of total percentage of S-phase of E2-treated cells, when compared with the group treated with E2 alone. However, unlike TCDD, both BaA and BaP stimulated G1-S-phase transition in cells treated with PAHs alone. This seems to suggest that both compounds exerted a dual type of effect on MCF-7 cells—while partially inhibiting cell cycle progression in cells treated with E2 as a strong mitogen, both compounds stimulated S-phase entry in cells synchronized by serum deprivation.
DISCUSSION
Perturbation of the balance between cell proliferation and apoptosis have been suggested to play a significant role in mediating and modifying the action of carcinogens in various target organs (Roberts et al., 1997). The increased proliferative rate may increase probability of initiated clonal cell expansion and increase the risk of DNA modification during its replication (Dipple et al., 1999; Klaunig et al., 2000). It has been hypothesized that PAHs, being both widespread environmental pollutants and important cigarette smoke constituents, could be involved in breast carcinogenesis as one of environmental risk factors (Burdick et al., 2003; Hecht, 2002). However, the available data on induction of cell proliferation by PAHs in breast epithelial cells are still not sufficient and often controversial, although this mode of action could contribute to tumor promotion. PAHs, or their hydroxylated metabolites, can act as estrogens in various estrogenicity assays (Charles et al., 2000; Clemons et al., 1998; Fertuck et al., 2001; Gozgit et al., 2004; Vondráek et al., 2002). Nevertheless, it has been suggested that only hydroxylated forms of PAHs are able to induce a transcription of endogenous ER-regulated genes (Gozgit et al., 2004), and some PAHs have been proposed to act as antiestrogens (Arcaro et al., 1999; Chaloupka et al., 1992), in a manner similar to other AhR ligands (Safe and Wrmke, 2003; Wang et al., 1998). Contrary to that, BaA can stimulate cell cycle progression in human breast carcinoma cells in an ER-dependent manner (Vondráek et al., 2002), and BaP quinones can activate EGFR in the ER-negative MCF-10A human breast epithelial cell line, which may also lead to induction of cell proliferation (Burdick et al., 2003). In the present study, we investigated effects of selected PAHs that have been previously shown to either directly or indirectly (by producing estrogenic metabolites) activate ER, on proliferation of human breast carcinoma MCF-7 cells, which express high levels of endogenous ER.
The major finding of this study was that both BaA and BaP induced proliferation of MCF-7 cells grown in estrogen-free medium, at concentrations as low as 100 nM. The third compound tested, Fla, was only a weak inducer of cell proliferation, increasing cell numbers by approximately 50% at 1 μM concentration (Figs. 1 and 2). Both BaA and BaP are carcinogenic compounds, listed as suspected human carcinogens (WHO, 1998). However, unlike BaP, BaA is a relatively weak mutagen, which produces a significantly lower amount of DNA-adducts than BaP in human cells (Binková and rám, 2004). Our data suggest that induction of cell proliferation by BaA was mediated by ER, because it was prevented by antiestrogen ICI 182,780. BaA was also found to increase DNA-synthesis and to promote G1-S-phase cell cycle transition in serum-deprived MCF-7 cells. Contrary to that, BaA did not induce p53 expression, confirming that it is only a weak genotoxin in MCF-7 cells. Taken together, the experimental evidence presented in this study seem to suggest that BaA can stimulate cell proliferation in estrogen-sensitive cells. This mechanism could contribute to its carcinogenic effects.
In contrast to BaA, BaP is a potent mutagen which is known to produce large amounts of BPDE-DNA adducts, which in turn leads to S-phase arrest/delay and induction of apoptosis (Binková and Sram, 2004; Chen et al., 2003; Jeffy et al., 1999; Khan and Dipple, 2000). BaP has been also suggested to produce quinone metabolites leading to induction of oxidative stress, associated both with oxidative DNA damage and transactivation of EGFR (Bolton et al., 2000; Burdick et al., 2003). In the present study, BaP induced S-phase arrest and at high concentrations also cell death, which is in accordance with the above studies. However, we found that BaP also stimulated proliferation of MCF-7 cells within concentration range 100 nM–1 μM. This proliferative effect was abolished by ICI 182,780. This is in accordance with a recent study showing that BaP can induce ER-dependent cell prolieration in vitro (Tsai et. al., 2004). Pifithrin-, which prevented induction of S-phase arrest, was found to potentiate cell proliferation induced by BaP (Fig. 5). It did not increase cell numbers in DBalP-treated group, suggesting that DBalP (a model genotoxic compound), in contrast to BaP, does not activate ER-driven processes in MCF-7 cells. A specific inhibitor of EGFR kinase had no effect either on BaP- or on BaA-stimulated cell proliferation, and EGFR was probably not involved in the observed stimulation of cell proliferation. Thus, our data showed that BaP can stimulate cell proliferation in MCF-7 cells by a mechanism involving ER activation, which is further potentiated by p53 inhibitor. Induction of cell proliferation and DNA synthesis could further contribute to the proposed "stealth properties" of BaP as a carcinogen, and to increase a likelihood of DNA replication on a damaged template (Dipple et al., 1999). Nevertheless, the present data also show that the genotoxic events leading to cell cycle arrest and/or cell death would decrease cell numbers, thus leading to overestimation of antiestrogenicity of mutagenic compounds, such as PAHs.
The antiestrogenicity of PAHs has been suggested to be associated with the activation of AhR (Chaloupka et al., 1992; Gozgit et al., 2004; Safe and Wrmke, 2003). Both BaA and BaP have been reported to inhibit foci development in MCF-7 cell cultures (Arcaro et al., 1999). This seems to be in contrast with our data, however, it should be mentioned that conditions used for cell growth in the present study were significantly different from the postconfluent cell model, and different experimental settings might be responsible for these discrepancies. Because AhR ligands, such as TCDD, are known to exert their anti-proliferative effects at least in part via inhibition of G1-S-phase transition (Wang et al., 1998), we compared the effects of model PAHs and TCDD on cell cycle progression in MCF-7 cells synchronized by serum deprivation. Using a combination of DNA staining and detection of BrdU incorporation, we found that like TCDD, BaP and BaA also partially inhibited induction of S-phase entry by E2. However, unlike TCDD, both BaP and BaA also stimulated G1-S-phase transition, when applied to serum-starved cells, albeit to a lesser extent than E2 itself. Interestingly, dibenzo[a,h]anthracene, a strong AhR ligand, which has been shown to be antiestrogenic in MCF-7 cells (Arcaro et al., 1999), had the same effect as TCDD both on the E2-treated and untreated cells (data not shown). These results seem to support the hypothesis that unlike other PAHs, both BaP and BaA, or their metabolites that are less efficient inducers of AhR-mediated activity, can activate ER and stimulate cell proliferation. Recently, 6-methyl-1,3,8-trichlorodibenzofuran, a weak agonist and a partial antagonist of AhR, which is currently being tested for its potential therapeutic use, has been found to activate ER and to stimulate MCF-7 cells proliferation, suggesting that this dual type of activity can be observed with some compounds (Pearce et al., 2004).
High levels of both BaP and BaA can be found in the respirable particulate matter of atmospheric pollution in urban environment. Both compounds were determined to be present at concentrations of several hundreds of ng per mg of extractable organic matter in urban air samples (Binková et al., 2003; Gábelová et al., 2004). Moreover, both compounds are important cigarette smoke constituents (Hoffmann et al., 2001), and they represent a significant part of carcinogenic PAHs found in environmental samples (Gábelová et al., 2004). Therefore, they might be considered to pose a potential threat to human health. Due to the rapid metabolization of PAHs, there is currently not sufficient information on levels of PAHs or their metabolites in tissues, such as human mammary tissue. Although there is presently no evidence that specific tobacco smoke carcinogens reach human mammary tissue, there is convincing evidence that mammary carcinogens in tobacco smoke are taken up and metabolized in smokers (Hecht, 2002). It should be noted, however, that exposure to BaP and BaA occurs simultaneously with exposure to high amounts of other PAHs, including both mutagens and efficient AhR ligands. This suggests that the proliferative effects of BaP and BaA could be modulated also by antiestrogenic or genotoxic effects of other pollutants present in complex environmental mixtures or in cigarette smoke.
In conclusion, we found that two PAHs, BaA and BaP, can stimulate proliferation of human breast carcinoma MCF-7 cells in ER-dependent manner. In contrast to TCDD, a model persistent AhR ligand, both BaP and BaA stimulated S-phase entry when applied to G1-synchronized MCF-7 cells. These results seem to suggest that inhibitory effects of BaP and BaA on cell proliferation as AhR ligands could be overcome by activation of ER. Unlike BaA, BaP induced p53 expression, a partial S-phase arrest and cell death at high concentrations. Pifithrin- abolished both S-phase arrest and cell death, and it potentiated the proliferative effect of BaP, suggesting that both genotoxic and nongenotoxic events may interact in effects of BaP on cell proliferation. The proliferative effects of both carcinogenic compounds might contribute to tumor promotion and, ultimately, carcinogenesis in estrogen-sensitive tissues. However, whether this phenomenon is restricted to the particular cell line studied here, or whether it is of in vivo relevance remains to be elucidated.
ACKNOWLEDGMENTS
Authors thank Prof. Ana M. Soto and Prof. Carlos Sonnenschein (Tufts University, Boston, MA) for providing MCF-7 cell line. This work was supported by the Czech Science Foundation (grants no. 525/01/D076 and 525/03/1527) and by the Research Plan of the Academy of Sciences of the Czech Republic No. Z5004920.
NOTES
1 These authors contributed equally to this work.
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