Department of Environmental Toxicology and the Center for Environmental Health Sciences, University of California, Davis, California 95616
Received September 12, 2000; accepted November 20, 2000
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ABSTRACT |
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Key Words: erbB-2/neu/HER-2; LNCaP; mitogen-activated protein kinase (MAPK); pesticides; prostate cancer; tyrosine kinases.
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INTRODUCTION |
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Environmental pollutants and dietary factors have been implicated in the incidence of prostate cancer (Giles and Ireland, 1997). The mechanisms by which environmental pollutants may initiate or promote prostate cancer are unknown. However, many environmental pollutants are known to interfere with hormonal regulation in hormone sensitive tissues (Colborn et al., 1993
; Davis et al., 1993
), which provides a framework for the action of endocrine disrupters in hormonal carcinogenesis (Huff et al., 1996
). Organochlorine insecticides and their metabolites have been shown to antagonize the androgen receptor (for example, Kelce et al., 1995). Several epidemiological studies have identified an association between pesticide exposure and elevated rates of prostate cancer in farm workers and pesticide applicators (Blair and Zahm, 1995
; Fleming et al., 1999
; Mills, 1998
; Morrison et al., 1993
; Schreinemachers et al., 1999
).
The oncogene erbB-2 codes for an epidermal growth factor (EGF)-receptor family tyrosine kinase (erbB-2, also known as HER-2 or c-neu) that is implicated in several types of cancer, including prostate and breast cancers. In the hormone-dependant prostate cancer cell line LNCaP, erbB-2 is overexpressed and functions in hormonally regulated mitotic signaling pathways (Meyers et al., 1996). Forced overexpression of erbB-2 kinase induces progression of prostate cancer cells in vitro, via the mitogen activated protein kinase (MAPK) signaling pathway (Yeh et al., 1999
). MAPK signaling can confer hormone independence via a process of ligand-independent activation of the androgen receptor (Craft et al., 1999
).
Our laboratory has previously reported increased erbB-2 kinase activity in hormone-responsive MCF-7 breast cancer cells following treatment by the organochlorine insecticides ß-hexa-chlorocyclohexane (ß-HCH), o,p'-dichlorodiphenyltrichloroethane (o,p'-DDT), and other chlorinated compounds (Enan and Matsumura, 1998; Hatakeyama and Matsumura, 1999
). In the present study we tested the ability of various pesticides to stimulate erbB-2 kinase activity in LNCaP cells and whether cellular proliferation was also stimulated. Test compounds were chosen based on the potential for human exposure (e.g., widespread use in agricultural practice and presence as low-level residue in food commodities), and as being representative of a range of chemical classes. Our results, together with results from MCF-7 breast cancer cells, indicate that stimulation of erbB-2 kinase activity by these compounds and subsequent cellular proliferation may be common phenomena in hormone-responsive cancer cells.
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MATERIALS AND METHODS |
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Cell culture.
LNCaP and PC-3 human prostate cancer cells were obtained from the American Type Culture Collection (ATCC; Rockville, MD) and grown in a 37°C, in 5% CO2 atmosphere. LNCaP cells were routinely maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM glutamine, penicillin (100 units/ml), streptomycin (100 units/ml) and amphotericin (0.25 µl/ml). PC-3 cells were routinely maintained in F-12 medium supplemented with 7% fetal bovine serum and glutamine, penicillin, streptomycin, and amphotericin, as above. In whole-cell chemical treatment experiments, fetal bovine serum in each of the respective media was heat-inactivated and treated with charcoal-dextran to remove lipophilic steroidal compounds. Cells were passaged by trypsin-EDTA treatment, and passage number was kept to less than 10 passages from ATCC stocks.
Preparation of postnuclear fraction.
Cells were grown to approximately 75% confluence in 100 mm diameter culture dishes. To prepare the postnuclear fraction, cells were rinsed twice in cold HEPES (50 mM, pH 7.5) and scraped in 1 ml homogenization buffer (50 mM HEPES pH 7.5, 1.5 mM KCl, 10 mM MgCl2, 2 mM DTT, 1 mM PMSF, 2 µg/ml aprotinin, 0.5 µg/ml leupeptin, 1 µg/ml pepstatin A). The cell suspension was homogenized in a Dounce apparatus and centrifuged 12,000 x g for 10 min at 4°C. The supernatant was recovered as the postnuclear fraction. Aliquots were snap-frozen in liquid nitrogen and stored at 80°C.
Chemical treatment of postnuclear fraction.
The postnuclear fraction was adjusted to 1.0 mg/ml protein concentration. Aliquots of 200 µl were dosed with test compound dissolved in ethanol (vehicle concentration 1%) and incubated 10 min at ambient temperature. In antibody blocking experiments, postnuclear fraction was incubated with anti-erbB2 antibody 9G6 (0.1 µg) for 15 min prior to chemical treatment.
Protein tyrosine kinase assay.
Treated postnuclear fraction (protein concentration 1.0 mg/ml) was divided into 20 µl aliquots and the kinase reaction initiated by adding 15 µl reaction buffer, to result in final concentrations of 10 mM MnCl2, 10 µM Na3VO4, 0.25 µCi -32P-ATP, and 1 µM radioinert ATP in 40 mM HEPES pH 7.5 containing 10 µg EY kinase substrate and 20 µg cellular protein. After incubation at 37°C for 5 min, the reaction was terminated by spotting 25 µl of the reaction mixture on phosphocellulose paper (Whatman P81). The paper was washed 3 times in phosphoric acid (85 mM), rinsed in acetone, and radioactivity measured by liquid scintillation counting.
MAP kinase activation assay.
LNCaP cells were grown to confluence in RPMI 1640 medium supplemented with 10% fetal bovine serum. The cells were switched to stripped media (RPMI 1640 medium supplemented with 10% charcoal-dextran-treated fetal bovine serum) at 24 h and again at 2 h prior to addition of test chemicals. Test chemicals (EGF, 1 nM; o,p'-DDT, 100 nM) were added in stripped medium and incubated 10 min. Nuclear extracts of the treated cells were prepared for immunoblot analysis of phospo p44/42 MAP kinase. Cells were disrupted in hypotonic buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, PMSF, DTT, aprotinin, leupeptin, pepstatin A) and centrifuged at 3300 x g for 15 min. The nuclear pellet was recovered and washed twice in SHSG buffer (hypotonic buffer, 0.25 M sucrose, 0.5 % Triton X-100, 8.5 % glycerol, protease inhibitors) and once in SHS buffer (SHSG buffer minus detergents). The washed nuclear pellet was suspended in one volume buffer C (20 mM HEPES pH 7.9, 25% glycerol, 0.42 M NaCl, 0.5 mM EDTA, protease inhibitors) and incubated for 30 min on ice. The nuclear suspension was centrifuged for 15 min on an Eppendorf microfuge at maximum speed, and the supernatant was saved as the nuclear extract. Protein concentrations were determined and adjusted to 1.0 µg/µl. Nuclear proteins (25 µg) were resolved on a 10% SDSPAGE gel and transferred to PVDF-plus membrane. The membrane was probed with anti-phospho p44/42 MAP kinase at 1:1000 dilution, followed by anti-rabbit IgG-HRP conjugate at 1:4000 dilution. Bands were visualized by chemiluminescence.
Proliferation assay.
Proliferation assays were conducted as described by Yeh et al. (1999). Cells were seeded into 24-well plates at 5 x 105 cells/well. The medium was changed to charcoal-dextran stripped medium after 24 h and test compound, dissolved in ethanol, was added. Control cells received vehicle only (ethanol). The treated cells were incubated for 72 h, the medium removed, and stripped medium containing MTT (thyazolyl blue, 1 mg/ml) was added. After 3-h incubation the medium was removed, and the formazan product solubilized in isopropanol. Absorbances were recorded at 540 nm.
Statistical analysis.
Experiments were run in triplicate or quadruplicate, and each experiment was repeated 2 or 3 times. Data from tyrosine kinase assays were compared by t-test of treated versus control samples. Proliferation assays were compared by one-way ANOVA. The criterion for significance was a p value < 0.05 for all comparisons.
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RESULTS |
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DISCUSSION |
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ErbB-2 stimulation by o,p'-DDT and ß-HCH was quantitatively greater in the prostate cell lines than was previously shown in the MCF-7 breast-cell line. These results in two distinct prostate cell lines indicate that the specific activation of this kinase is not unique to the MCF-7 system but may be a common phenomenon in erbB-2-expressing cancer cells. This specific interaction of 2 organochlorine pesticides with this important mitotic signaling protein led us next to determine if other pesticides could cause this same type of stimulation. It must be noted that we did not intend to perform a structure-activity relationship study, but rather a succinct survey of major-use pesticides, to determine the range of agrochemicals having the potential to stimulate erbB-2 in the same manner as o,p'-DDT and ß-HCH. Of the compounds tested, permethrin (a pyrethroid insecticide), chlorothalonil (a chlorothalonitrile fungicide) and heptachlor epoxide (an organochlorine insecticide metabolite) also stimulated erbB-2 kinase activity under cell-free conditions.
Given that erbB-2 is a major mitogenic signal protein in prostate-cancer cells, it was anticipated that whole-cell treatment with erbB-2 stimulating compounds would affect cellular proliferation. Interestingly, while erbB-2 was activated by chemical treatment in both the hormone-dependent LNCaP and hormone-independent PC-3 cell lines, increases in cellular proliferation was observed only in LNCaP. This suggests that the proliferative effect due to o,p'-DDT is somehow dependent upon the androgen receptor-signaling pathway, and consequently raises the question of whether the androgen receptor is directly stimulated. It has been shown previously that o,p'-DDT can competitively displace androgen from its receptor (Kelce et al., 1995) with an IC50 of approximately 100 µM. However, the effects we observed on erbB-2 activation occurred at 100 nM (3 orders of magnitude difference). Therefore erbB-2 kinase activation may be the more sensitive cellular response related to cellular proliferation. In support of this conclusion, we were able to show that the potent antiandrogen p,p'-DDE (IC50 = 5 µM), a direct antagonist of the androgen receptor (Kelce et al., 1995
) could not block the proliferative effect of o,p'-DDT on LNCaP cells. The 20-fold difference in IC50s precludes significant displacement of p,p'-DDE by o,p'-DDT at the androgen receptor. Therefore the proliferative effect of o,p'-DDT in the presence of p,p'-DDE demonstrates that o,p'-DDT does not interact directly with the androgen receptor.
ErbB-2 kinase is known to activate unliganded androgen receptor in prostate cells (Craft et al., 1999). This is considered to represent one of many examples of "ligand independent activation" of hormone receptors (Weigel and Zhang, 1998
). In a number of cases, the modulation of growth factor signal-mediating kinase or phosphatase activities are known to lead to activation of hormone receptors. In the case of extensively studied breast cancer cell lines, such as MCF-7, the activation of MAPK p42/44 by the growth factor receptor erbB-2 is the critical event mediating subsequent activation of the unliganded estrogen receptor. Although less is known about the mechanism of ligand-independent activation of the androgen receptor in prostate cancer cells, the relationship between MAPK activation and androgen receptor action has been observed in hormone-independent prostate cancer cell proliferation (Abreu-Martin et al., 1999
). Androgen receptor target genes have been shown to be activated in the absence of androgen via an erbB-2/MAPK signal cascade (Yeh et al., 1999
; Zhu and Liu, 1997
). In LNCaP cells, heregulin, a natural activator of erbB-2 kinase (via the erbB-3 receptor) clearly activates MAPK (Grasso et al., 1997
). In this cell line, erbB-2 is synergized by low levels of androgen. This evidence, and our experimental results, support our hypothesis that, a priori, the activation of the erbB-2 kinase signaling pathway induced by certain pesticides should elicit ligand-independent activation of the androgen receptor via activation of MAPK, leading to cellular proliferation in androgen receptor-rich LNCaP cells.
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ACKNOWLEDGMENTS |
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NOTES |
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The contents of this report are solely the responsibility of the authors and do not necessarily represent the official views of the NIEHS, NIH.
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