Induction of cyclooxygenase expression and enhancement of malignant cell transformation by 2,3,7,8-tetrachlorodibenzo- p-dioxin

Detlef Wölfle2, Stefan Marotzki, Dorothee Dartsch, Wolfgang Schäfer1 and Hans Marquardt

Department of Toxicology, University of Hamburg Medical School, and Department of Toxicology and Environmental Medicine of the Fraunhofer Society, D-20146 Hamburg and
1 Department of Obstetrics and Gynecology, Medical School University of Freiburg, D-79106 Freiburg, Germany


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The potential role of arachidonic acid metabolism in the enhancement (promotion) of malignant transformation of C3H/M2 mouse fibroblasts by the tumor promoter 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) was investigated using inhibitors of cyclooxygenase and lipoxygenase activities. The promoting effects of TCDD (1.5 pM) and of the reference tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA; 0.4 mM) on carcinogen (N-methyl-N'-nitro-N-nitrosoguanidine or 3-methylcholanthrene)-pre-treated fibroblasts was abolished by cotreatment with indomethacin, hydrocortisone, caffeic acid or nordihydroguaiaretic acid. A differential inhibition was found with N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide, a selective inhibitor of the cyclooxygenase isoenzyme COX-2: the promoting effect of TPA, but not that of TCDD, was abolished. Therefore, the role of the cyclooxygenase isoenzymes COX-1 and COX-2 during chronic exposure to TCDD was studied in more detail. Long-term treatment with TCDD (4–7 weeks) induced the expression of COX-1 and COX-2 mRNA in C3H/M2 fibroblasts (up to 2-fold). The enhanced expression of COX-2 protein in TCDD-treated fibroblasts was confirmed by western blot analysis. Concomitantly, the accumulation of the prostaglandins (PGs) PGE2 and 6-keto-PGF1{alpha}, which were identified as major metabolites of arachidonic acid in C3H/M2 cell cultures, was enhanced (~2-fold) following long-term treatment with TCDD (0.15 and 1.5 pM). The results suggest that the stimulation of arachidonic acid metabolism caused by a sustained cyclooxygenase induction is a critical event in the promoting action of TCDD in mouse fibroblasts in vitro. However, in contrast to TPA, the TCDD-mediated enhancement of malignant cell transformation may not specifically depend on the induction of COX-2 but, additionally, the induction of COX-1 activity may be necessary.

Abbreviations: COX-1/2, cyclooxygenase-1/2; EET, epoxyeicosatrienoic acid; GAPDH, glycerol aldehyde phosphate dehydrogenase; HETE, hydroxyeicosatetraenoic acid; MCA, 3-methylcholanthrene; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; NDGA, nordihydroguaiaretic acid; NS-398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide; NSAID, non-steroidal anti-inflammatory drug; PG, prostaglandin; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TPA, 12-O-tetradecanoylphorbol-13-acetate.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototype of many halogenated aromatic hydrocarbons, is a ubiquitous, persistent environmental contaminant and the most powerful tumor promoter in rodent bioassays (1). Increasing epidemiological evidence suggests that TCDD exposure may cause a higher incidence of various types of human cancer (2). The toxic effects of TCDD including carcinogenicity, teratogenicity, immunosuppression, dermal and liver toxicity are thought to be mediated by a cytosolic protein, the Ah (aryl hydrocarbon) receptor. However, the molecular mechanisms leading to tumor promotion have not been elucidated. TCDD has been reported to modulate the expression of a variety of proteins involved in the regulation of growth and differentiation, e.g. growth factors (3), protein kinases (4,5) and products of protooncogenes (6). Given the pivotal role of these factors in tumor promotion it is important to elucidate the action of TCDD on growth and differentiation-specific signal transfer pathways. Indirect evidence for the involvement of signalling molecules, e.g. reactive oxygen species and metabolites of arachidonic acid, is the inhibitory effect of antioxidants and anti-inflammatory agents on promoting effects of TCDD (7) and of various other tumor promoters such as 12-O-tetradecanoylphorbol-13-acetate (TPA) (8,9). The anticarcinogenic effect of many naturally occurring agents, including vitamins and flavones, may be due to their antioxidant properties and their inhibitory effect on arachidonic acid metabolism (10).

The role of arachidonic acid metabolites as modulators in the multi-step process of carcinogenesis, particularly in tumor promotion, has often been postulated with supportive evidence from epidemiological and experimental studies. This notion is strengthened by reports on a reduced mortality and a lower incidence of human colon cancer following chronic consumption of non-steroidal anti-inflammatory drugs (NSAIDs), e.g. acetylsalicylic acid (11). Sulindac (12) and indomethacin (13) suppress the number and size of colonic polyps in patients with familial adenomatous polyposis. In accord with the human data, animal studies have shown an anti-carcinogenic action of NSAIDs on gastrointestinal and other tumors (1417). Moreover, inhibitors of cyclooxygenase (18) and lipoxygenase (19) activities decrease the tumor promoting effects of various structurally unrelated agents and inhibit the growth of tumor cells in vivo (14) and in vitro (20). The impact of arachidonic metabolism on tumorigenesis is further strongly suggested by the following observations: (i) the levels of specific metabolites of arachidonic acid and the expression of cyclooxygenases and lipoxygenases are enhanced in various human and rodent tumors (9,2123); (ii) the cyclooxygenase isoenzyme COX-2 is induced in normal tissues by the treatment with tumor promoters, e.g. TCDD (2426) and TPA (9). Interestingly, the induction of COX-2 has been observed in pathological processes including tumor promotion (27). Thus, in a quest for a more mechanistic risk assessment pertaining to tumor promoters such as dioxin-like compounds, the expression of cyclooxygenase activities may provide an important link necessary for this goal.

Recently, we have studied the mechanisms of tumor promotion by TCDD in the two-step transformation assay with C3H/M2 mouse fibroblasts using antioxidants (7). In the present study, hydrocortisone, inhibitors of cyclooxygenases [i.e. indomethacin and N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide (NS-398)] and inhibitors of lipoxygenases [i.e. caffeic acid and nordihydroguaiaretic acid (NDGA)], were used to investigate the role of arachidonic acid metabolism in the enhancement (promotion) of the transformation of carcinogen-pre-treated fibroblasts induced by TCDD. Additionally, the formation of arachinonic acid metabolites and the induction of the cyclooxygenase isoenzymes COX-1 and COX-2 were studied. The data show that chronic TCDD treatment results in a sustained enhancement of prostaglandin (PG) formation caused by the induction of COX-1/COX-2 mRNA and protein expression in mouse fibroblasts.


    Materials and methods
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 Abstract
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 Materials and methods
 Results
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Chemicals
TCDD was obtained from Ökometric (Bayreuth, Germany) and was >99% pure. The following compounds were purchased from the indicated companies: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), TPA, indomethacin and calcium ionophor A 23187 from Sigma (Deisenhofen, Germany); 3-methylcholanthrene (MCA) from Eastman (Organic Chemicals, Rochester, NY); NDGA and NS-398 from BIOMOL (Hamburg, Germany); basal Eagle's medium and fetal calf serum were purchased from Gibco BRL (Eggenstein, Germany); [3H]arachidonic acid from DuPont NEN (Dreieich, Germany); non-radioactive eicosanoids from Cayman (Ann Arbor, USA); and HPLC grade solvents from Merck (Darmstadt, Germany). All reagents for PCR analysis and western blotting were obtained from Sigma or Merck, if not indicated otherwise.

Malignant transformation of C3H/M2 mouse fibroblasts
This assay was carried out as described previously (28) and adjusted to measure tumor promotion according to procedures described (29). Briefly, cells harvested from logarithmically growing stock cultures (between passages 5 and 20) were plated on day 0 in basal Eagle's medium supplemented with 10% fetal calf serum into 60 mm dishes to determine their plating efficiency (100 cells/dish) and the transformation rate (1000 cells/dish). After 24 h, the cultures were treated for 24 h with initiating agents, i.e. MNNG (0.1 µg/ml), MCA (1 µg/ml) or solvent control, dimethyl sulfoxide (DMSO; 0.5%). Thereafter, the medium was renewed and the cells were allowed to grow in fresh medium. Beginning on day 5 until the end of the experiment, with each of the (twice weekly) medium renewals the tumor promoters TCDD (1.5 pM) and TPA (0.25 µg/ml), or solvent, were added. The media were further supplemented with hydrocortisone, indomethacin, NS-398, caffeic acid or NDGA at non-toxic concentrations, as indicated. The cells were fixed and stained after 2 weeks (to determine their plating efficiency) or after 8 weeks (to determine the transformation rate). Cells from selected cultures were harvested for PCR and western blot analyses. Concomitantly, medium samples were taken for HPLC analysis and enzyme immunoassays.

HPLC analysis
C3H/M2 fibroblasts pretreated with MNNG or MCA were labeled with [3H]arachidonic acid (8 µCi/dish) for 16 h at the end of the promoting treatment with TCDD or TPA. Thereafter, release of 3H label into the medium (0.5–1 h) in the presence of the calcium ionophor A23187 (5 µM) was determined by liquid scintillation counting. Aliquots of supernatants of culture media were frozen at –80°C. Media were analyzed by reverse-phase HPLC with radioactivity detection as described previously (30). Briefly, 500 µl aliquots of centrifuged media were injected and the pre-column (µBondpak C18/Corasil; Waters, Eschborn, Germany) was flushed with water–methanol–phosphoric acid (85:15:0.02) at a flow rate of 2.5 ml/min for 3 min. After column switching, eicosanoids were transferred to a Novapak C18 column (Waters, Eschborn, Germany) which was kept at 40°C. As mobile phase, solvent A (water–actetonitrile–phosphoric acid; 72:28:0.02) and solvent B (water–actetonitrile–phosphoric acid; 5:95:0.02) were applied. From 0–13 min, elution was carried out isocratically with 100% of solvent A. Between 13 and 52 min, solvent B was increased to 80%, and in a final step to 100% at 57 min. The flow rate was 1 ml/min. Radioactivity detection was performed with a radiomonitor LB 405 (Bertold, Wildbad, Germany). Peak identification was controlled daily with tritiated eicosanoid standards. Retention times of the eicosanoids not available as radioactive standards were determined in separate HPLC runs with UV detection.

Enzyme immunoassay (EIA) analysis
For the quantitative analysis of PG concentrations by EIA kits (Cayman, Ann Arbor, MI) aliquots of the supernatants of culture media were taken 24 h after medium renewal at different times after the beginning of TCDD treatment. The aliquots were diluted 1:10 in fresh culture medium and the assays were conducted according to the instructions of the supplier.

Semiquantitative RT–PCR
Total RNA was isolated from fibroblast cultures by the method of Chomczynski and Sacchi (31) and RT–PCR was performed as described previously (32). Briefly, RNA (0.8 µg) was reverse transcribed into cDNA using Superscript II RNase H-RT (Life Technologies) according to the instructions of the manufacturer. One percent of the cDNA reaction was added to a 100 µl PCR reaction that contained 200 µM dNTPs, 2 mM MgCl2, 1 U PrimeZyme DNA Polymerase (Biometra, Göttingen, Germany) in 1x Ampli-Buffer (10 mM Tris–HCl pH 8.8 at 25°C, 50 mM KCl, 0.1% Triton X-100) and 0.3 µM of each primer. For efficient PCR amplification the primers were selected to yield products in the size range of 130–230 bp. The sequences of the primers were: 5'-CCATGGAGAAGGCTGGGG-3' and 5'-CTAAGCAGTTGGTGGTGC-3' for glycerol aldehyde phosphate dehydrogenase (GAPDH; 195 bp); 5'-CAAAAGAACCCAGTGTCC-3' and 5'-ATGAGTCCATCTGTTCCC-3' for cyclooxygenase (COX)-1 (133 bp); 5'-CACAGTATGATGTAACAGTCC-3' and 5'-AACACAGCTACGAAAACC-3' for COX-2 (224 bp). After the addition of mineral oil, an initial denaturation step was performed for 3 min at 94°C and cDNA was amplified under the following reaction conditions: 34 cycles for 15 s at 94°C, 30 s at 54°C and 1 min at 72°C. During the exponential phase of amplification, an aliquot of 10 µl was taken from each of the last seven cycles. PCR products were visualized by UV light after agarose gel electrophoresis and ethidium bromide staining. Using the digital image the optical density (OD) of each band was measured (One Dscan, Version 1.0, Scanalytics). The OD of each COX-1 and COX-2 band was normalized by the corresponding GAPDH band.

Western blot analysis
Cells (3–4x106) were harvested, washed with PBS containing protease inhibitors (16 µg/ml benzamidine–HCl, 10 µg/ml phenanthroline, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 100 mM phenylmethylsulfonyl fluoride), resuspended in a mixture of PAGE buffer (62.5 mM Tris–HCl pH 6.8, 1% SDS, 10% glycerol, 5% mercaptoethanol, 0.5 mM EDTA pH 7.2, bromophenol blue) and 2x Laemmli buffer (120 mM Tris–HCl pH 6.8, 4% SDS, 200 mM dithiothreitol, 30% glycerol, 0.6% Na-EDTA, bromophenol blue), again supplemented with the protease inhibitors, and sonicated. Equal amounts of protein (50 µg) were heated to 95°C for 5 min and separated on a 10% acrylamide–bisacrylamide minigel containing 0.1% SDS. The proteins were then blotted onto a nitrocellulose membrane from Bio-Rad (Munich, Germany). After blocking in Tris-buffered saline containing 0.01% Tween-20 and 5% skimmed milk powder, sequential incubation with goat polyclonal anti-COX-2 IgG (1:500; Santa Cruz, Heidelberg, Germany) and HRP conjugated anti-goat IgG (1:10 000; Santa Cruz, Heidelberg, Germany) was carried out. The investigated proteins were detected with the ECL-System from Amersham Buchler (Braunschweig, Germany) on X-ray films (Fujifilm) and digitalized using the Gel print2000i system (v.2.3) from BioPhotonics Corp. (Ann Arbor, MI).

Statistics
Statistical significance was calculated with Student's t-test for unpaired observations. A value of P < 0.05 was considered significant.


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Inhibition of TCDD-mediated promotion of cell transformation by inhibitors of arachidonic acid metabolism
Hydrocortisone, an inhibitor of phospholipase A2 activity and cyclooxygenase induction, or indomethacin, an inhibitor of cyclooxygenase activity, were used at non-toxic concentrations to examine the possible role of metabolites of arachidonic acid in the process of malignant cell transformation. The phorbol ester TPA was included as a standard tumor promoter in our experiments (Table IGo); the promoting effect of TPA was reported to be inhibited by indomethacin in vivo (9) and in vitro (33). The malignant transformation of C3H/M2 cells induced by maximally effective concentrations of the initiating agents MNNG (0.5 µg/ml) or MCA (10 µg/ml) was unaffected by hydrocortisone or indomethacin (data not shown). However, hydrocortisone reduced the initiating effect of low concentrations of MNNG (0.1 µg/ml) and MCA (1 µg/ml) and abolished the promoting effect of TPA and TCDD (Table IGo). Similarly, indomethacin (20 µg/ml) abolished the promotion by TPA and TCDD. Moreover, a 10-fold lower concentration of indomethacin which had no effect on the initiating action of a low concentration of MCA (1 µg/ml) was sufficient to prevent the effects of both tumor promoters, TPA and TCDD (Table IGo). In contrast, NS-398, a specific inhibitor of the cyclooxygenase isoenzyme COX-2, abolished the promoting effect of TPA but not that of TCDD (Table IGo).


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Table I. Inhibition of tumor promoter-induced enhancement of C3H/M2 fibroblast transformation by inhibitors of cyclooxygenase and 5-lipoxygenase activities
 
To investigate the role of the lipoxygenase pathway, two inhibitors of 5-lipoxygenase, i.e. NDGA (Table IGo) and caffeic acid (data not shown), were used. These inhibitors also prevented the promotion by TPA and TCDD following pre-treatment with MCA.

The effect of TCDD on arachidonic acid release
MCA-pre-treated C3H/M2 fibroblasts were labeled with [3H]arachidonic acid at the end of the promoting treatment with TPA and TCDD. Thereafter, [3H]arachidonic acid release into the medium was measured in the presence of the calcium ionophor A23187: In TCDD (1 pM)-treated cultures the arachidonic acid release was only slightly enhanced (1.3–1.5-fold) as compared with control cultures. The arachidonic acid metabolites from the culture medium were separated by HPLC analysis (Figure 1Go). No significant qualitative changes in the elution profiles of TPA-, TCDD- or MCA (24 h)- and TCDD (7 weeks)-treated cultures compared with controls were observed. The major peaks were identified as the PGs 6-keto-PGF1{alpha} and PGE2 and a minor peak represented PGF2{alpha}. Further peaks were attributed to hydroxyeicosatetraenoic acid (HETE) which was found following treatment with TPA or TCDD and to epoxyeicosatrienoic acid (EET) which was found following TCDD-treatment.



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Fig. 1. Typical reverse-phase HPLC elution profile of arachidonic acid metabolites released by C3H/M2 fibroblasts. Fibroblasts were treated with (A) the solvent DMSO (0.5%), (B) TPA (0.25 µg/ml), (C) TCDD (1.5 pM) or (D) pre-treated with MCA (1 µg/ml) and, thereafter, treated with TCDD. At the end of the long-term treatment (7 weeks) with TCDD or TPA, fibroblasts were labeled with [3H]arachidonic acid for 24 h. 3H-labeled metabolites released into the medium in the presence of the calcium ionophor A23187 were analyzed by HPLC as described in Materials and methods. Peaks were identified as the PGs 6-keto-PGF1{alpha}, PGF2{alpha} and PGE2, HETE and EET.

 
TCDD-mediated stimulation of PG release
Quantitative changes in the concentrations of 6-keto-PGF1{alpha} and PGE2 in the culture medium after TCDD treatment were analyzed by competitive enzyme immunoassays. After short-term (24 h) treatment of C3H/M2 fibroblasts with TCDD, no significant differences in PG concentrations were detected. However, after long-term treatment (7 weeks), TCDD markedly enhanced the levels of PGE2 in MCA-initiated but not in control cultures (Table IIGo). This effect of TCDD was observed at very low TCDD concentrations (0.15 and 1.5 pM) which were also maximally effective in promotion of the malignant cell transformation (Table IIGo). The TCDD effect was time-dependent and significant in MCA-pre-treated fibroblast cultures at ~3 weeks after the beginning of TCDD exposure (Figure 2Go). Similarly, an enhancement of 6-keto-PGF1{alpha} accumulation by TCDD was observed (data not shown). Addition of indomethacin (2 µg/ml) to the cultures inhibited the effect of TCDD and reduced the PG levels to <10% of those in control cultures.


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Table II. Enhancement of PGE2 accumulation after TCDD treatment for 7 weeks
 


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Fig. 2. Time-dependent enhancement of PGE2 release from MCA-pre-treated C3H/M2 fibroblasts during tumor promoter treatment with TCDD. The cultures were treated with TCDD (1.5 pM) for the indicated time periods. Thereafter, the medium was renewed and 24 h later aliquots of the medium supernatants were collected for EIA analysis. Data represent means ± SD (duplicate cultures from three independent experiments). *P < 0.05; as compared with MCA-pre-treated cultures without TCDD.

 
Induction of cyclooxygenase expression by TCDD
The expression of COX-1 and COX-2 mRNA in TCDD-treated C3H/M2 fibroblast cultures was analyzed by RT–PCR. A transient, dose-dependent induction of COX-1 mRNA (1.2–1.6-fold) and COX-2 mRNA (1.4–1.9-fold) was found at 24 h after addition of TCDD (0.1–100 pM). This short-term induction by TCDD was slower and less pronounced than the TPA (0.4 mM)-mediated induction of COX-2 mRNA (maximum induction at 6 h: 2.3-fold; data not shown). However, a sustained induction of COX-1 and COX-2 mRNA was observed after long-term treatment (4 weeks) with TCDD (1.5 pM) in MCA-pre-treated cultures, but not in control (DMSO) cultures (Table IIIGo). These findings are in accordance with the data on PG accumulation (Table IIGo; Figure 2Go) in MCA-pre-treated fibroblasts. At the end of the transformation experiments (7 weeks with TCDD at 1.5 and 150 pM), a dose-dependent induction of COX-1 mRNA (1.2–1.6-fold) and COX-2 mRNA (1.4–1.9-fold) was found in DMSO-, MNNG- or MCA-pre-treated cultures. Similarly, the expression of COX-2 protein was induced by TCDD (1.5 and 150 pM) and by TPA at the end of the transformation experiments (Figure 3Go).


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Table III. Induction of COX-1 and COX-2 mRNA expression by long-term treatment of C3H/M2 fibroblasts with TCDD (4 weeks)
 


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Fig. 3. Dose-dependent enhancement of COX-2 protein in C3H/M2 fibroblasts after long-term treatment with TCDD. Control (A1–A4) or MCA-pre-treated cells (C1–C4) were cultured for 6 weeks with DMSO (A1, C1), TPA (A2, C2) or TCDD [1.5 pM (A3, C3) or 150 pM (A4, C4)]. At the end of the transformation experiment cells were harvested for western blot analysis.

 

    Discussion
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Inhibitors of arachidonic acid metabolism have been proposed as chemopreventive agents at different stages of the multi-step process of carcinogenesis, most importantly in the reversible, long-term phase of tumor promotion. In the present study, using C3H/M2 mouse fibroblasts pre-treated with the initiating agents MNNG and MCA, we have found a complete inhibition of TCDD- and TPA-induced promotion of cell transformation by inhibitors of arachidonic acid metabolism. Glucocorticoids, which decrease both basal and induced levels of COX-2 expression (34), and indomethacin, an inhibitor of cyclooxygenase (preferentially COX-1) activity, are potent preventive agents of TPA-induced skin tumor promotion (35) and promotion of BALB/3T3 transformation in vitro (33). Furthermore, indomethacin has been reported to inhibit tumor promotion by non-phorbol ester compounds (3638) in vivo and in vitro. In accordance with these observations, hydrocortisone and indomethacin at non-toxic concentrations abolished TPA-mediated promotion in the transformation assay with MCA- and MNNG-pre-treated C3H/M2 cells (Table IGo). We extend the knowledge on the anti-promoting effect of hydrocortisone and indomethacin to TCDD as a prototype of polyhalogenated aromatic tumor promoters. Recently, we have found that indomethacin also prevents the promoting effect of polychlorinated biphenyls, i.e. the dioxin-like 3,3',4,4'-tetrachlorobiphenyl and the non-dioxin-like 2,2',4,4',5,5'-hexachlorobiphenyl (unpublished data). In conclusion, inhibition of cyclooxygenases may be a more general chemopreventive strategy to suppress the tumor promoting actions of structurally unrelated compounds.

In pathogenic processes including tumor promotion, COX-2 has been suggested to play a critical role (39,40). COX-2 expression is rapidly induced by growth factors (41,42), cytokines (43), TPA (44), TCDD (2426) and other tumor promoters (45,46). Constitutive overexpression of COX-2 has been reported in human (21,22,44,47,48) and animal tumors (9,23), in cell lines corresponding to different stages of tumor development (9) and in transformed, tumorigenic cell lines (49). In accordance with these data, we have found a significantly enhanced expression of COX-2 mRNA in chemically transformed C3H/M2 fibroblasts isolated from malignant foci (~2-fold; unpublished data) and of COX-2 mRNA (up to 2-fold) (32) and protein (Figure 3Go) in normal fibroblasts following long-term treatment with TCDD (6 weeks). In addition to the induction of COX-2 mRNA, the levels of COX-1 mRNA were also enhanced by TCDD in MCA-pre-treated cultures (Table IIIGo). These results indicate that the long-term treatment with TCDD does not specifically induce the expression of COX-2. Given the cell- and tissue-specific induction of COX-2 by TCDD (25,26) the implication of COX-1 activity in pathological processes, e.g. inflammation (50) and possibly tumor promotion, should not be underestimated.

Selective COX-2 inhibitors have an anti-carcinogenic activity in rat colon tumorigenesis (51). However, in the present study using NS-398 as a selective COX-2 inhibitor, we observed an inhibition of TPA- but not of TCDD-induced promotion of C3H/M2 transformation. This unexpected result might be explained by the fact that COX-1 induction in C3H/M2 cells contributes to the promoting action of TCDD. Additionally, the differential inhibition by NS-398 may be due to TPA- and TCDD-mediated signal transduction via agent-specific cellular receptors (6,52) and nuclear response elements (24). The induction by TCDD of the COX-2 expression (Figure 3Go; Table IIIGo) (53) and of the PGE2 accumulation in C3H/M2 fibroblast cultures (Figure 2Go) has been found with different kinetics and to a lower extent compared with the induction by TPA. Similar results were obtained for other non-phorbol ester tumor promoters in comparison with TPA (46). However, for any evaluation of COX-2 expression and PG formation in vitro one has to consider that there might be only very few (initiated?) cells per dish which are triggered to increase arachidonic acid metabolism. Therefore, the enhanced PG formation by the induced cells might be balanced by the metabolic activity of the bulk of normal cells. The enhancement of PGE2 accumulation was apparently more pronounced in cultures with transformed foci than in cultures without foci. These findings may be explained by a differential expression of COX-2 and PG formation in single fibroblasts during long-term treatment with tumor promoters. Currently, we are addressing this question by in situ RT–PCR to determine COX-2 expression in single cells of TCDD-treated cultures.

A substantial database implicates PGs, e.g. PGF2{alpha}, as key regulators in tumor cell growth and carcinogenesis, particularly in tumor promotion. In mouse skin tumor promotion by TPA, the indomethacin-mediated suppression of papilloma formation was reversed by simultaneous treatment with PGF2{alpha} rather than with PGE2 (9,35). We obtained similar results on TPA-induced promotion of C3H/M2 transformation showing that PGF2{alpha} reverses the inhibition by NS-398 (unpublished data). Moreover, preliminary data indicate that the addition of PGF2{alpha} alone may enhance the transformation of carcinogen-pre-treated C3H/M2 fibroblasts. However, a number of studies suggest PG-independent mechanisms of chemoprevention by NSAIDs (27,54). Several NSAIDs have targets other than cyclooxygenases, e.g. cAMP-dependent protein kinase (55), superoxide anion generation by NADPH oxidase, phospholipase C (27) and glutathione transferase (56). Moreover, a modulation of tumor promoter-associated processes, i.e. apoptosis (57) and intercellular communication (58), by NSAIDs has been reported. Thus, the exact relationship of cyclooxygenase inhibition and tumor prevention by NSAIDs remains uncertain.

In addition to cyclooxygenases, lipoxygenase isoenzymes might be involved in tumor development and tumor promotion by TPA (59,60). It has been suggested that induction of 8-lipoxygenase activity is an essential feature of the hyperplastic and tumor promoting activity of TPA (61). Following TPA- and TCDD-treament of C3H/M2 mouse fibroblasts, lipoxygenase products, i.e. HETEs, were detected in the culture medium (Figure 1Go). Moreover, lipoxygenase inhibitors are potent chemo-preventive agents (62) that inhibit tumor promotion by TPA (19) and other promoters (63). Similarly, in the C3H/M2 transformation assay the lipoxygenase inhibitors caffeic acid and NDGA abolished the promoting effect of TPA and TCDD (Table IGo). However, it cannot be ruled out that the anti-tumor promoting effect of these lipoxygenase inhibitors are at least partly due to their antioxidative effects. It has been suggested that NDGA inhibits the TPA-mediated promotion by scavenging lipid hydroperoxides and decreasing the oxidative metabolism of arachidonic acid (35). Furthermore, the prevention of liver tumor formation by acetylsalicylic acid was also associated with an inhibition of oxidative processes in hepatocarcinogenesis (64). Recently, we have shown that antioxidants are potent inhibitors of the TPA- and TCDD-promotion in the C3H/M2 transformation assay (7). Several lines of evidence indicate an association between arachidonic acid metabolism and the formation of reactive oxygen species: (i) lipid peroxidation and H2O2 activate phospholipase A2 (65,66); (ii) various peroxides trigger via cyclooxygenase- or lipoxygenase-dependent pathways oxidative stress-induced events (66,67); and (iii) arachidonic acid metabolism leads to the formation of oxygen species (64).

In summary, chronic treatment of mouse fibroblasts with TCDD at a very low concentration (1.5 pM) induces the expression of COX-1 and COX-2 and eventually PG formation. This activation of arachidonic acid metabolism is associated with the TCDD-mediated enhancement of malignant cell transformation. Moreover, we have shown that inhibitors of arachidonic acid metabolism abolish the promoting effects of structurally different tumor promoters (e.g. TPA and TCDD) on the enhancement of fibroblast transformation. Even though our understanding of the anti-tumor promoting mechanisms of cyclooxygenase and lipoxygenase inhibitors is not yet complete, it is reasonable to speculate that these compounds, particularly those which are dietary components (68), have important implications for human health. Thus, further studies are warranted to elucidate the dose-, compound-, species- and tissue-specific effects of anti-inflammatory agents (69,70) and to provide a sound scientific basis for a rational chemoprevention of cancer.


    Acknowledgments
 
The authors are grateful to E.Becker, U.Hamel, A.Piasecki and A.Ruge for excellent technical assistance and to K.Werner for performing the HPLC runs. The work was funded by grants of the Bundesministerium für Bildung, Forschung und Technologie (07 DIX12 A1).


    Notes
 
2 To whom correspondence should be addressed at: Federal Institute for Health Protection of Consumers and Veterinary Medicine (BgVV), FG 703, Thielallee 88-92, D-14195 Berlin, Germany Email: d.woelfle{at}bgvv.de Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received July 14, 1999; revised September 24, 1999; accepted September 30, 1999.