During the cooking of meat, mutagenic and carcinogenic heterocyclic amines are formed, the most abundant of which, 2-amino-1-methyl-6-phenylimidazo[4-5-b]pyridine (PhIP), induces tumors of the prostate, colon, and mammary gland in rats. Humans consuming cooked meat are exposed to PhIP on a daily basis, yet few studies have assessed the effects of PhIP at dietary relevant concentrations. In addition to its genotoxic properties, recent studies have shown that PhIP can activate estrogen receptor–mediated signaling pathways at doses that are similar to those that may be present in the body following consumption of a cooked meat meal. In the present study, we examined whether such doses of PhIP can affect estrogen receptor–independent signal transduction via the mitogen-activated protein kinase (MAPK) extracellular signal–related kinase (ERK) pathway to influence proliferation and migration in the human mammary epithelial cell line MCF10A and the prostate cancer cell line PC-3. At doses shown to have a proliferative effect on MCF10A cells (10−11–10−7 mol/L), PhIP induced a rapid, transient increase in phosphorylation of both MAPK/ERK kinase 1/2 and ERKs. Inhibition of this pathway significantly reduced the PhIP-induced proliferation of MCF10A cells and the migration of PC-3 cells. The data presented here show that levels of PhIP that approximate to human dietary exposure stimulate cellular signaling pathways and result in increased growth and migration, processes linked to the promotion and progression of neoplastic disease. These findings provide strong evidence that PhIP acts as a tumor initiator and promoter and that dietary exposure to this compound could contribute to carcinogenesis in humans. [Cancer Res 2007;67(23):11455–61]

Of the environmental factors linked with human carcinogenesis, diet is regarded as a major determinant, in particular the consumption of meat (1). During the cooking of meat and fish, a number of mutagenic compounds known as heterocyclic amines are formed, and these have been shown to be carcinogenic in rodent bioassays (2, 3). The most abundant of these compounds is 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), which has been shown to induce tumors of the breast, prostate and colon in animal models (4, 5). The finding that PhIP induces cancer in three tissue sites where tumors occur with high incidence in the Western world and the presence of PhIP in a Western-style diet rich in cooked meat suggest that this compound may pose a significant threat to human health. In support of this, PhIP-DNA adducts have been detected in human breast, colon, and prostate tissue (69), whereas a number of epidemiology studies have reported associations between consumption of red meat, PhIP intake, and cancer of the breast, prostate, and colon (1013). It is generally acknowledged that the carcinogenic potential of PhIP is initiated with the formation of PhIP-DNA adducts resulting in mutation. The mechanistic basis of this involves primarily CYP1A2-mediated oxidation to the N-hydroxy derivative (14), then esterification resulting in an unstable product that generates a nitrenium ion that attacks and adducts to guanine in DNA (15).

In addition to its genotoxic properties, there is a growing body of evidence suggesting that PhIP also has effects on mammary gland development, proliferation, and signal transduction that could contribute to carcinogenesis. In rat models, PhIP exposure is associated with increased cell proliferation in terminal end buds of the mammary gland and inhibition of the differentiation of these terminal end buds into alveolar buds, which are structures that are less susceptible to carcinogenesis (16). PhIP retards the involution of the mammary gland in lactating rats on weaning through the inhibition of apoptosis of the secretory mammary epithelial cells (17). In addition, micromolar concentrations of PhIP have been shown to have a direct inhibitory effect on apoptosis in the human mammary epithelial cell line MCF10A (18), and this was associated with activation of the mitogen-activated protein kinase (MAPK) extracellular signal–regulated kinase (ERK) pathway, which plays a critical role in the regulation of cell proliferation, differentiation, and survival.

Recent work in our laboratory has shown that at nanomolar concentrations, which is thought to be close to circulating plasma levels following a cooked meat meal, PhIP stimulates estrogen receptor α–induced activation of estrogenic genes and induces a proliferative response in estrogen-dependent MCF-7 breast cancer cells (19). In these studies, PhIP was also found to activate ERK1 and ERK2, and this was only partially inhibited by the antiestrogen ICI 182,780, thus suggesting that PhIP may also be able to affect cell signaling pathways by estrogen receptor–independent mechanisms.

Estrogens play a crucial role in regulating the growth, differentiation, and function of many reproductive tissues, and the principal physiologic estrogen 17β-estradiol is considered to be a major risk factor in the development of breast cancer and has also been implicated in the etiology of colorectal and prostate cancer (2022). A major role of the MAPK ERK pathway is to modulate gene transcription involved in cell proliferation and migration, and ERK activation has been reported in human mammary and prostate cancer tissues (23, 24). Given that consumption of cooked meat can lead to low levels of PhIP being present in body fluids such as breast milk (25), it is possible that PhIP may exert estrogenic and mitogenic effects in humans that could contribute to carcinogenesis.

To investigate whether low doses of PhIP (nanomolar concentrations) can directly affect MAPK signaling, we have used MCF10A cells, which are estrogen receptor negative, and a human prostate cancer cell line, PC-3.

Materials

MCF10A human mammary epithelial cells were purchased from the American Type Culture Collection. Human prostate adenocarcinoma PC-3 cells were obtained from the European Collection of Cell Cultures. Ham's F-12/DMEM (1:1) growth medium, horse serum, l-glutamine, penicillin/streptomycin, epidermal growth factor (EGF), insulin, and trypsin-EDTA were purchased from Invitrogen Corporation. Cholera toxin was purchased from Merck Biosciences Ltd. PhIP (MW, 224) was purchased from Toronto Research Chemicals, Inc. CellTiter-Blue cell viability assay was from Promega. Inhibitors of EGF receptor (EGFR; Tyrphostin AG1478) and insulin-like growth factor (IGF)-I receptor (IGF-IR; I-OMe-AG538) were purchased from Merck Biosciences. SDS-PAGE molecular weight standards were obtained from Bio-Rad. Hybond nitrocellulose membrane and Hyperfilm autoradiography film were from Amersham Biosciences. BCA protein assay kit and SuperSignal West Pico chemiluminescent substrates were purchased from Perbio Science UK Ltd. Mouse monoclonal anti–phosphorylated ERK1/ERK2 (Tyr204) antibody and rabbit polyclonal anti–phosphorylated and anti–unphosphorylated ERK1/ERK2 antibodies were from Santa Cruz Biotechnology, Inc. Rabbit polyclonal anti–phosphorylated MAPK/ERK kinase (MEK)-1/MEK2 (Ser217/221) and total MEK1/MEK2 antibodies were from Cell Signaling Technology. Western blot stripping solution and QCM Chemotaxis 96-well cell migration assay were from Chemicon International. p44/42 MAPK assay kit was purchased from Cell Signaling Technology. All other chemicals and reagents were purchased from Sigma-Aldrich.

Cell Proliferation Assay

MCF10A cells. MCF10A cells were routinely cultured in 1:1 Ham's F-12/DMEM supplemented with 5% horse serum, 2 mmol/L l-glutamine, 100 IU/mL penicillin/100 μg/mL streptomycin, 20 ng/mL EGF, 100 ng/mL cholera toxin, 0.01 mg/mL insulin, and 500 ng/mL hydrocortisone. Cells were cultured in an incubator maintained at 5% CO2 and 37°C. A serum-free medium was prepared containing all supplements except horse serum (IHE medium). For experiments, MCF10A cells were seeded into six-well plates (2.5 × 104 per well) and allowed to adhere overnight. The following day, medium was removed and cells were washed with PBS and then treated with PhIP in IHE medium in the presence or absence of the MEK1/2 inhibitor PD98059 (50 μmol/L). Cells were cultured under treatment for 6 days and the medium was replaced every 2 days. Dilutions of PhIP were made up in absolute ethanol whereas inhibitors were made up in DMSO, and all compounds were administered at a final concentration of 0.1% vehicle in the media.

Following treatment, viability was measured using the CellTiter-Blue cell viability assay. Six hundred microliters of CellTiter-Blue reagent were added to each well (containing 3 mL of medium), and the cells were then incubated at 37°C for a further 2 h. The assay is based on the reduction of resazurin (7-hydroxy-3H-phenoxazin-3-one-10-oxide) to resorufin, and preliminary experiments showed a direct correlation between fluorescence and MCF10A cell number (data not shown). Following incubation, resorufin fluorescence was measured with a fluorimeter (BMG PolarStar) at 560-nm excitation and 590-nm emission. All experiments were done in triplicate wells and repeated on three separate occasions. The percent proliferative potency was calculated as described previously (19).

PC-3 cells. PC-3 cells were routinely cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 100 IU/mL penicillin/100 μg/mL streptomycin in an incubator maintained at 5% CO2, 37°C.

For proliferation experiments, cells were seeded into flat-bottomed 96-well plates at a density of 2,500 per well and allowed to adhere overnight. The following day, medium was removed from the wells and cells were rinsed briefly with sterile PBS before addition of serum-free medium (RPMI 1640 supplemented with penicillin/streptomycin). After incubation in serum-free medium for 24 h, the medium was replaced with RPMI 1640 supplemented with 0.5% FBS and antibiotics, and cells were treated with varying concentrations of PhIP or ethanol solvent control in low-serum (0.5%) medium. Cells were treated for 6 days in total, with chemicals and media replaced every 2 days.

Immunoblotting

MCF10A or PC-3 cells were seeded in T150 flasks and grown to 75% confluency. Cells were then washed twice with PBS and placed in serum-free medium devoid of all supplements and antibiotics for 24 h. Various concentrations of PhIP or ethanol solvent control were then added to the cells. After treatment, cells were washed twice with ice-cold PBS and then lysed on ice for 5 min in lysis buffer consisting of 50 mmol/L Tris-HCl (pH 8.5), 150 mmol/L NaCl, 1% NP40, and 5 mmol/L EDTA, supplemented with 10 mmol/L sodium pyrophosphate, 5 mmol/L sodium orthovanadate, 50 μg/mL phenylmethylsulfonyl fluoride, 20 μg/mL aprotinin, and 10 μg/mL leupeptin. Cells were then scraped from the flasks and centrifuged at 14,000 × g at 4°C for 10 min. Supernatants were collected and protein content was determined with a Pierce BCA protein assay kit.

Proteins were separated by SDS-PAGE (10% gels) and transferred onto a nitrocellulose membrane. Following incubation with primary and secondary antibodies, proteins were visualized using an enhanced chemiluminescence detection system.

MAPK Assay

To investigate the activation of ERK1 and ERK2 by PhIP, a kinase assay was carried out according to the manufacturer's protocol (Cell Signaling Technology). The assay is based on the immunoprecipitation of active MAPK (ERK1/ERK2) from cell lysates, followed by incubation of the immunoprecipitate with an ELK-1 fusion protein in the presence of ATP and kinase buffer; under these conditions, immunoprecipitated active ERK1/ERK2 phosphorylates ELK-1.

MCF10A cells were seeded into flasks and allowed to adhere overnight. The following day, cells were washed and placed in serum-free medium devoid of all supplements and were treated 24 h later with PhIP in the presence or absence of inhibitors. All inhibitors were added to cells 30 min before PhIP treatment.

Following treatment, cell lysis, immunoprecipitation, and kinase assay were done according to the manufacturer's instructions and phosphorylated ELK-1 was visualized by immunoblotting.

Cell Migration/Chemotaxis Assay

A cell migration assay was carried out with PC-3 or MCF10A cells using a chemotaxis assay (Chemicon International). Briefly, PC-3 or MCF10A cells were incubated in their respective serum-free medium for 24 h, following which they were detached from flasks, suspended in quenching medium (serum-free medium containing 5% bovine serum albumin) and PhIP or ethanol, and seeded into Boyden migration chamber inserts placed in a 96-well plate. The inserts contain a microporous membrane with an 8-μm pore size. Inserts were placed over wells containing serum-free media plus chemoattractant (10% FBS). After a 24-h treatment period, cells/media were discarded from the top side of the migration chamber insert and the chamber was placed in the wells of a new 96-well plate containing cell detachment solution. Following incubation for 30 min at 37 C, the insert was then discarded, and a solution of lysis buffer and CyQuant GR dye was added to each well. CyQuant is a green fluorescent dye that exhibits strong enhancement of fluorescence when bound to cellular nucleic acids released by the lysis buffer, enabling assessment of the relative number of migrated cells. Fluorescence was determined with a fluorimeter at 480/520 nm.

Cell Proliferation

The ability of PhIP to influence proliferation in the estrogen receptor–negative breast epithelial cell line MCF10A was examined by a fluorometric assay as described in Materials and Methods. Under serum-free conditions, MCF10A is dependent on addition of exogenous EGF and insulin for proliferation. Insulin acts through IGF-IR. After 6 days of treatment in medium devoid of serum but containing EGF, insulin, and other supplements (IHE medium), low doses of PhIP (10−9–10−7 mol/L) caused a weak increase in cell proliferation in MCF10A cells (Fig. 1). Concurrent treatment with the MAPK pathway inhibitor PD98059 significantly abolished the increase in cell proliferation induced by PhIP, as did the RAF-1 kinase inhibitor I (Fig. 1).

Figure 1.

The effect of PhIP on proliferation of MCF10A cells (A) and PC-3 cells (B). Cells were treated with various concentrations of PhIP for 6 d in low-serum (0.5%) medium. Columns, mean of three separate experiments each done with between three and eight replicates; bars, SD. In some experiments, the MEK1/2 inhibitor PD98059 (PD; 50 μmol/L) or the RAF-1 kinase inhibitor I (Raf I; 10−7 mol/L) was added. **, P < 0.01; *, P < 0.05, compared with ethanol (EtOH) control; #, P < 0.01, compared with treatment with PhIP or ethanol alone (ANOVA followed by Dunnett multiple comparisons test).

Figure 1.

The effect of PhIP on proliferation of MCF10A cells (A) and PC-3 cells (B). Cells were treated with various concentrations of PhIP for 6 d in low-serum (0.5%) medium. Columns, mean of three separate experiments each done with between three and eight replicates; bars, SD. In some experiments, the MEK1/2 inhibitor PD98059 (PD; 50 μmol/L) or the RAF-1 kinase inhibitor I (Raf I; 10−7 mol/L) was added. **, P < 0.01; *, P < 0.05, compared with ethanol (EtOH) control; #, P < 0.01, compared with treatment with PhIP or ethanol alone (ANOVA followed by Dunnett multiple comparisons test).

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Low concentrations of PhIP (10−12–10−8 mol/L) also stimulated the proliferation of PC-3 cells grown under low-serum conditions (Fig. 1). The effect of PhIP on proliferation seemed to be more potent in the prostate cell line than in MCF10A cells, reaching an ∼80% increase in proliferation versus controls in PC-3 compared with a maximal increase in proliferation of ∼20% in MCF10A.

Cell Migration/Chemotaxis

Addition of PhIP to PC-3 cells in the upper compartment of the Boyden migration chamber significantly increased the ability of the cells to migrate through the pores to the chemotactic (FBS) stimulus compared with the vehicle-treated control cell population (Fig. 2). This effect was seen at similar levels with all concentrations of PhIP examined (10−12–10−7 mol/L), with the migratory ability of cells exposed to PhIP increasing from ∼2-fold over cells treated with ethanol alone. In MCF10A cells, 10−10 mol/L PhIP seemed to slightly elevate migration; however, the effect was not significant and decreased with increasing PhIP concentration (Fig. 2).

Figure 2.

Effect of PhIP on migration of MCF10A cells (A) and PC-3 cells (B). Cells were placed in the upper compartment of the migration chamber in serum-free medium containing PhIP or ethanol for 24 h. Cells that had migrated to the underside of the chamber were detached from the membrane, lysed, and quantitated by fluorescent staining with CyQuant dye. In some experiments, the MEK1/2 inhibitor PD98059 was added (50 mmol/L). Columns, mean of three separate experiments each done in five replicate wells; bars, SE. **, P < 0.01, compared with ethanol control; #, P < 0.01, compared with PhIP 10−9 mol/L alone (ANOVA followed by Dunnett multiple comparisons test).

Figure 2.

Effect of PhIP on migration of MCF10A cells (A) and PC-3 cells (B). Cells were placed in the upper compartment of the migration chamber in serum-free medium containing PhIP or ethanol for 24 h. Cells that had migrated to the underside of the chamber were detached from the membrane, lysed, and quantitated by fluorescent staining with CyQuant dye. In some experiments, the MEK1/2 inhibitor PD98059 was added (50 mmol/L). Columns, mean of three separate experiments each done in five replicate wells; bars, SE. **, P < 0.01, compared with ethanol control; #, P < 0.01, compared with PhIP 10−9 mol/L alone (ANOVA followed by Dunnett multiple comparisons test).

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PD98059 was added to PC-3 cells to examine the involvement of the MAPK pathway in PhIP-induced chemotaxis. As shown in Fig. 2, PD98059 (50 μmol/L) significantly inhibited the number of cells migrating through the chamber in response to PhIP treatment, implying the involvement of the MAPK pathway in mediating PhIP-induced PC-3 cell migration.

MAPK Pathway

Phosphorylation of MAPK pathway proteins. Having observed an increase in cell proliferation and migration that was ablated by the MAPK ERK pathway inhibitor PD98059, the effect of PhIP on this pathway was further examined. This signaling pathway plays a major role in the regulation of cell proliferation and differentiation (26) and involves a protein kinase cascade following growth factor stimulation, which leads to the successive phosphorylation, and thus activation, of RAF-1, MEK1/2, and then ERK1 and ERK2, which then target a number of transcription factors involved in regulating mitogenesis, such as ELK-1. PD98059 acts by inhibiting MEK1/2.

In MCF10A, PhIP rapidly induced a small but significant increase in the phosphorylation of MEK1/2 in the first hour following treatment at doses (10−7–10−9 mol/L) associated with increased proliferation (Fig. 3). Total levels of MEK1/2 were unchanged during this period and effectively served as a loading standard. A similar effect was observed for the phosphorylation of ERK1/2 (Fig. 4), confirming that PhIP was indeed activating MEK1/2 to induce phosphorylation of the ERK protein. This increased phosphorylation of both MEK1/2 and ERK1/2 protein persisted for at least 60 min (Figs. 3 and 4), but was no longer apparent 4 h after initial exposure (data not shown). Similar effects were noted in PC-3 cells (Figs. 3 and 4) with lower concentrations of PhIP (10−9–10−11 mol/L PhIP). In PC-3 cells, PhIP-mediated phosphorylation of MEK1/2 was shorter lived, being attenuated after 10 min. However, phosphorylation of ERK1/2 remained elevated at 60 min, particularly at the lower dose (10−11 mol/L PhIP). Thus, PC-3 cells seemed to be more sensitive to PhIP-mediated MAPK activation than were the MCF10A cells. This is consistent with the proliferation and migration characteristics of the two cell lines.

Figure 3.

The effect of PhIP on the phosphorylation status of MEK1/2 in MCF10A and PC-3 cells. A, MCF10A cells. Phosphorylated and total forms of MEK1/2 were evaluated by immunoblotting with protein extracts obtained from MCF10A cells treated with PhIP in serum-free medium for the indicated time periods. 1, ethanol; 2, 10−7 mol/L PhIP; 3, 10−9 mol/L PhIP. B, blots were quantified with a Kodak Image Station and Kodak ID Image Analysis Software. Columns, mean of three separate blots; bars, SE. *, P < 0.05; **, P < 0.01, compared with respective DMSO control (ANOVA with Tukey-Kramer posttest). C, PC-3 cells. Phosphorylated and total forms of MEK1/2 were evaluated by immunoblotting with protein extracts obtained from PC-3 cells treated with PhIP in serum-free medium for the time periods indicated. 1, ethanol; 1, 10−9 mol/L PhIP; 3, 10−11 mol/L PhIP. D, blots were quantified with a Kodak Image Station and Kodak ID Image Analysis Software. Columns, mean of three separate blots; bars, SE. *, P < 0.05; **, P < 0.01, compared with respective DMSO control (ANOVA with Tukey-Kramer posttest).

Figure 3.

The effect of PhIP on the phosphorylation status of MEK1/2 in MCF10A and PC-3 cells. A, MCF10A cells. Phosphorylated and total forms of MEK1/2 were evaluated by immunoblotting with protein extracts obtained from MCF10A cells treated with PhIP in serum-free medium for the indicated time periods. 1, ethanol; 2, 10−7 mol/L PhIP; 3, 10−9 mol/L PhIP. B, blots were quantified with a Kodak Image Station and Kodak ID Image Analysis Software. Columns, mean of three separate blots; bars, SE. *, P < 0.05; **, P < 0.01, compared with respective DMSO control (ANOVA with Tukey-Kramer posttest). C, PC-3 cells. Phosphorylated and total forms of MEK1/2 were evaluated by immunoblotting with protein extracts obtained from PC-3 cells treated with PhIP in serum-free medium for the time periods indicated. 1, ethanol; 1, 10−9 mol/L PhIP; 3, 10−11 mol/L PhIP. D, blots were quantified with a Kodak Image Station and Kodak ID Image Analysis Software. Columns, mean of three separate blots; bars, SE. *, P < 0.05; **, P < 0.01, compared with respective DMSO control (ANOVA with Tukey-Kramer posttest).

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Figure 4.

The effect of PhIP on the phosphorylation status of ERK1 and ERK2 in MCF10A and PC-3 cells. A, MCF10A cells. Phosphorylated and total forms of ERK1/2 were evaluated by immunoblotting with protein extracts obtained from MCF10A cells treated with PhIP in serum-free medium for the indicated time periods. 1, ethanol; 2, 10−7 mol/L PhIP; 3, 10−9 mol/L PhIP. B, blots were quantified with a Kodak Image Station and Kodak ID Image Analysis Software. Columns, mean of three separate blots; bars, SE. *, P < 0.05; **, P < 0.01, compared with respective DMSO control (ANOVA with Tukey-Kramer posttest). C, PC-3 cells. Phosphorylated and total forms of ERK1/2 were evaluated by immunoblotting with protein extracts obtained from PC-3 cells treated with PhIP in serum-free medium for the time periods indicated. 1, ethanol; 1, 10−9 mol/L PhIP; 3, 10−11 mol/L PhIP. D, blots were quantified with a Kodak Image Station and Kodak ID Image Analysis Software. Columns, mean of three separate blots; bars, SE. *, P < 0.05; **, P < 0.01, compared with respective DMSO control (ANOVA with Tukey-Kramer posttest).

Figure 4.

The effect of PhIP on the phosphorylation status of ERK1 and ERK2 in MCF10A and PC-3 cells. A, MCF10A cells. Phosphorylated and total forms of ERK1/2 were evaluated by immunoblotting with protein extracts obtained from MCF10A cells treated with PhIP in serum-free medium for the indicated time periods. 1, ethanol; 2, 10−7 mol/L PhIP; 3, 10−9 mol/L PhIP. B, blots were quantified with a Kodak Image Station and Kodak ID Image Analysis Software. Columns, mean of three separate blots; bars, SE. *, P < 0.05; **, P < 0.01, compared with respective DMSO control (ANOVA with Tukey-Kramer posttest). C, PC-3 cells. Phosphorylated and total forms of ERK1/2 were evaluated by immunoblotting with protein extracts obtained from PC-3 cells treated with PhIP in serum-free medium for the time periods indicated. 1, ethanol; 1, 10−9 mol/L PhIP; 3, 10−11 mol/L PhIP. D, blots were quantified with a Kodak Image Station and Kodak ID Image Analysis Software. Columns, mean of three separate blots; bars, SE. *, P < 0.05; **, P < 0.01, compared with respective DMSO control (ANOVA with Tukey-Kramer posttest).

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MAPK assay. To further confirm the finding that PhIP stimulates the MAPK pathway, a kinase assay was carried out with MCF10A cells. The assay involved evaluation of the amount of phosphate incorporated into an ELK-1 fusion protein that serves as a substrate for immunoprecipitated active ERK1/2. This assay confirmed that ERK1 and ERK2 are activated within 30 min of PhIP treatment. This activation was observed at concentrations as low as 10−12 mol/L PhIP (Fig. 5).

Figure 5.

Effect of PhIP on the kinase activity of ERK1 and ERK2 in MCF10A cells. ERK1/2 kinase activity was determined by the detection of ELK-1 phosphorylation by active ERK1 and ERK2 immunoprecipitated from MCF10A cells treated with PhIP for 30 min in serum-free medium (A), in the presence of the MEK1/2 inhibitor PD98059 (50 μmol/L; B), EGFR tyrosine kinase inhibitor AG1478 (AG; 30 nmol/L; C), or IGF-IR kinase inhibitor I-OMe-AG538 (30 μmol/L; D) for 30 min in serum-free medium. Bands represent separate kinase assays carried out with different cell lysates.

Figure 5.

Effect of PhIP on the kinase activity of ERK1 and ERK2 in MCF10A cells. ERK1/2 kinase activity was determined by the detection of ELK-1 phosphorylation by active ERK1 and ERK2 immunoprecipitated from MCF10A cells treated with PhIP for 30 min in serum-free medium (A), in the presence of the MEK1/2 inhibitor PD98059 (50 μmol/L; B), EGFR tyrosine kinase inhibitor AG1478 (AG; 30 nmol/L; C), or IGF-IR kinase inhibitor I-OMe-AG538 (30 μmol/L; D) for 30 min in serum-free medium. Bands represent separate kinase assays carried out with different cell lysates.

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MCF10A cells were also treated in the presence of inhibitors of the MAPK pathway to gain an insight into the mechanism by which PhIP activates ERK1/2. As expected, the MEK1/2 inhibitor PD98059 dramatically reduced the kinase activity of ERK1/2 induced by PhIP (Fig. 5). This is consistent with the effect of PD98059 on MCF10A proliferation (Fig. 1). AG1478, a highly specific inhibitor of the EGFR tyrosine kinase, also produced a marked suppression of kinase activity (Fig. 5), as did I-OMe-AG538, an inhibitor of IGF-IR kinase (Fig. 5). The effect of this latter inhibitor was less marked than that of PD98059 or AG1478.

Finally, to support our finding that the MEK1/2 inhibitor PD98059 dramatically reduced the migration of PC-3 cells in response to PhIP treatment (Fig. 2), we examined the phosphorylation of ERK1/2 in the presence of PD98059. The result shown in Fig. 6 confirmed that phosphorylation of ERK1/2 induced by PhIP was significantly attenuated.

Figure 6.

The effect of PhIP on the phosphorylation status of ERK1 and ERK2 in PC-3 cells in the presence of the MEK1/2 inhibitor PD98059 (50 μmol/L). Phosphorylated and total forms of ERK1/2 were evaluated by immunoblotting with protein extracts obtained from PC-3 cells treated with PhIP and PD98059 in serum-free medium.

Figure 6.

The effect of PhIP on the phosphorylation status of ERK1 and ERK2 in PC-3 cells in the presence of the MEK1/2 inhibitor PD98059 (50 μmol/L). Phosphorylated and total forms of ERK1/2 were evaluated by immunoblotting with protein extracts obtained from PC-3 cells treated with PhIP and PD98059 in serum-free medium.

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Few studies have assessed the effects of PhIP at concentrations that are likely to be present in humans following consumption of a cooked meat meal. Human consumption of heterocyclic amines has been estimated as ranging from as low as 1 μg to more than 50 μg per day (27). Given the rapid and extensive bioavailability of PhIP, circulating plasma levels are likely to be in the nanomolar range following consumption of cooked meat (28). At such concentrations, the genotoxicity of PhIP is not detectable but its ability to activate signaling pathways is (19). The data presented here show that extraordinarily low doses of PhIP are able to increase proliferation and stimulate the MAPK signaling pathway in an estrogen receptor–negative human mammary epithelial cell line, MCF10A, and the prostate cancer cell line PC-3.

Previous studies have shown that high doses of PhIP (10−4 mol/L) could inhibit apoptosis in MCF10A cells under conditions of serum and growth factor deprivation, an in vitro model for apoptosis of the involuting mammary gland (18). The experimental conditions for the proliferation studies described here involved the withdrawal of serum but not exogenous growth factors. These growth factors (EGF and IGF-I) continue to exert prosurvival effects (29), and as such, the increased cell viability observed in these studies following the addition of PhIP is not believed to be an effect of apoptosis inhibition but is due to an increase in mitogenic stimulation. In concordance with this, PhIP treatment also resulted in increased proliferation in PC-3 cells in low-serum medium. Indeed, the effects of PhIP seemed to be more profound in PC-3 cells. It is possible that the effect was less marked in the MCF10A cell model because the additional stimulation provided by PhIP was only marginal in the presence of the added exogenous cell survival factors.

The development of migratory and invasive properties is a key event in the oncogenic progression of cells (30), and cell motility is one of the rate-limiting steps of invasion. Chemotactic motility, which involves the sensing of a concentration gradient of chemoattractants such as cytokines or growth factors, reorganization of the actin cytoskeleton, and subsequent movement toward the chemoattractant, is therefore believed to play a major role in tumor invasion and metastasis (31).

Using a cell migration assay, it was shown that PhIP was able to significantly increase the number of migratory PC-3 cells. This stimulatory effect of PhIP was even more potent than its mitogenic effects, with cell movement more than doubling at doses as low as 10−11 mol/L. In MCF10A cells, however, PhIP had no significant effect on cell migration, although a very slight increase was observed with the two lowest doses used. Unlike PC-3 cells, which are derived from a prostate carcinoma bone metastasis and already display invasive properties, MCF10A cells are not transformed and have the phenotypic characteristics of normal breast cells (32). Whereas the fully transformed, malignant breast epithelial cell line MCF-7 seems to be constantly motile on laminin-5–coated surfaces, the majority of MCF10A cells maintain a stationary phenotype in such a model (33). Our findings suggest that PhIP is better able to promote motility in cells that are predisposed to an invasive phenotype.

The ERK signal transduction cascade plays a central role in cell proliferation and motility (34, 35). These proliferative and migratory effects influence tumor promotion and progression, and ERK signaling is stimulated by a number of tumor promoters including 12-O-tetradecanoylphorbol-13-acetate (36). Elevated ERK has been reported in human breast tumors (23, 37) whereas changes in several key regulatory pathways that can lead to ERK activation, such as ErbB, protein kinase C, and insulin-like growth factor receptor, are frequently elevated in breast tumors and are associated with resistance to hormonal therapy (3841). Levels of activated ERK have also been shown to be significantly increased in human prostate tumors compared with benign samples, and activation has been correlated with increasing Gleason score (24, 42).

Given the importance of ERK1 and ERK2 signaling in cell proliferation, motility, and carcinogenesis, we examined whether this pathway might be associated with the mitogenic and migratory effects of PhIP. Pretreatment of MCF10A cells with the highly specific MEK inhibitor PD98059 led to a dramatic reduction in the proliferation of MCF10A cells. This is to be expected because both EGF and IGF-I activate the ERK pathway to stimulate proliferation (43, 44). The increased proliferation over control cells induced by PhIP was also completely abolished by PD98059, suggesting that PhIP is stimulating proliferation in these cells by an ERK-mediated mechanism. Activation of the ERK MAPK pathway also seemed to be critical for the effect of PhIP on PC-3 cell migration because addition of the MEK1/2 inhibitor PD98059 almost entirely ablated the increase in motility stimulated by PhIP. This correlates with a previous work showing the critical role played by this pathway in PC-3 cell migration and anchorage-independent growth (45).

The effect of PhIP on the phosphorylation of MEK1/2 and ERK1/2 was examined in both MCF10A and PC-3 cells. At doses shown to have a proliferative effect on MCF10A cells (10−11–10−7 mol/L), PhIP induced a rapid, transient increase in phosphorylation of both MEK1/2 and ERKs. This increase was observed at time points up to an hour after PhIP exposure, and levels of phosphorylation had returned to basal 4 h after treatment, a pattern of activation similar to that observed following mitogenic stimulation through the addition of growth factors such as EGF (46).

Kinase assays were also carried out in MCF10A cells to confirm that PhIP-induced phosphorylation of ERKs led to activation of ERK1/2 by measuring the ability of immunoprecipitated activated ERKs to phosphorylate the downstream transcription factor ELK-1. PhIP (10−7 mol/L) was shown to increase the level of ELK-1 phosphorylation, and as expected, this was reduced by the MEK inhibitor PD98059. Notably, PhIP was able to increase ERK activity at concentrations as low as 10−10 and 10−12 mol/L.

The mechanism by which PhIP stimulates the ERK pathway is as yet unclear. PhIP has previously been found to have estrogenic properties (19); however, the cell lines used in the present experiment are estrogen receptor negative. A number of xenoestrogens have been shown to be able to modify estrogen receptor–independent signaling pathways. For example, p-nonylphenol can increase ERK1 and ERK2 activation in bovine adrenal medulla cells in an estrogen receptor–independent manner (47) whereas the phytoestrogens genistein and quercetin have been shown to activate ERK1 and ERK2 via transactivation of EGFR in a process mediated by the G protein–coupled receptor homologue GPR30 (48). Inhibitors of both EGFR and IGF-IR were therefore used in an attempt to shed light on how PhIP is able to activate the MAPK pathway. Interestingly, the highly specific inhibitor of EGFR kinase, AG1478, produced a marked inhibition of the kinase activity of ERKs induced by PhIP, as measured by kinase assay, whereas I-OMe-AG538, an inhibitor of IGF-IR kinase, also reduced PhIP-induced MAPK activity, although not by as great an extent. These results raise the possibility that PhIP activates the ERK pathway by modulating the activation of growth factor receptors, specifically EGFR and IGF-IR.

The genotoxicity of PhIP has generally been assumed to be the major mode of action by which it induces cancer; however, this study and others suggest that alternative mechanisms may also be important. Whereas PhIP-DNA adducts have been found in the target organs of the compound in chronic feeding studies (5, 49), they have also been found to be distributed in other nontarget tissues such as the pancreas and lungs without induction of tumors (50). It is therefore likely that PhIP possesses additional mechanisms of action that may influence its carcinogenic effects.

Previous work in our group and other laboratories has shown that at low levels (10−6–10−10 mol/L), PhIP is able to stimulate proliferation in estrogen receptor–positive human mammary carcinoma MCF-7 cells in a manner that is inhibited by the antiestrogen ICI 182,780 (19, 51, 52). More recently, Nakai et al. (53) reported that whereas PhIP treatment of Big Blue rats resulted in increased mutation frequencies in all lobes of the prostate, increased proliferation was only observed in the ventral lobe, the area where PhIP specifically induces prostate cancer. The authors concluded that in addition to acting as a tumor initiator, PhIP may also act as an organ- or lobe-specific promoter.

PhIP-DNA adducts have been reported in the human mammary gland, colon, and prostate, supporting the concept that PhIP has initiating potential in humans (79). The results reported here that low-dose PhIP is able to stimulate mammary and prostate cell proliferation and motility of prostate cancer cells provide further evidence to suggest that PhIP also has the potential to exert a promotional influence on cells previously initiated by PhIP or other genotoxic agents. An increase in the rate of cell proliferation will reduce the time available for DNA repair processes to remove DNA adducts before cell replication converts adducts to mutations, thereby resulting in a higher probability of induction of mutations.

One of the requirements of tumor promotion is repeated exposure to the promoting agent. A small proportion of a dose of PhIP remains unmetabolized and is excreted in the urine for at least 12 h following consumption of a cooked meat meal (28). This suggests that cells are exposed to low levels of PhIP for prolonged periods, and regular intake of cooked meat would be expected to provide repeated, prolonged exposure to both PhIP and PhIP metabolites. Meat-consuming humans are likely to be exposed to low levels of PhIP on a daily basis. Whereas CYP1A2-mediated metabolic activation of PhIP to genotoxic species has been a major area of study, other biological activities of PhIP have not been examined. The genotoxic potency of PhIP is well established, but is only detectable at comparatively high doses of the chemical (>10−5 mol/L). We have described an important biological activity of the chemical at remarkably low doses, in some instances ∼10−12 mol/L, which are well below the Km (54) of xenobiotic metabolizing enzymes (∼10−5 mol/L). It is concentrations such as these that are likely to persist in humans consuming cooked meat. It is therefore possible that dietary PhIP could act both as a tumor initiator and as a promoter and that exposure to this compound may contribute to carcinogenesis in humans.

Grant support: U.K. Food Standards Agency and a scholarship from Imperial College London (S.K. Creton).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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