Introduction

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Several epidemiological studies have demonstrated an association between the long-term consumption of ASA and a reduced risk of colon cancer (Giovannucci et al., 1995; Greenberg et al., 1993; Rosenberg et al., 1991; Thun et al., 1991). Although the mechanism for reduction of colorectal cancer by ASA is not clear, ASA and other NSAID have been shown to be potent inhibitors of tumor formation in rodent models of chemically induced colon cancer (Reddy et al., 1992). Furthermore, treatment of FAP patients with NSAID results in a reduction in the number and size of adenomas present in the large intestine (Giardello et al., 1993).

Aspirin and other NSAID directly target PGHS (Vane, 1971). ASA irreversibly inhibits PGHS by acetylation of serine-530 thereby excluding access for arachidonic acid (Loll et al., 1995). PGHS is a key enzyme in the production of PG, prostacyclins and thromboxanes (Piomelli, 1993; Smith, 1989).

Increased PG production by tumors has been associated with aggressive tumor progression (Honn et al., 1981), and inhibition of PG synthesis has resulted in growth retardation of tumors in experimental animals (Lupulescu, 1978). However, other authors have reported that PG are not involved in the antitumor (Alberts et al., 1995) or in the cytostatic action of NSAID (DeMello et al., 1980).

Different mechanisms have been proposed to explain the antitumorogenic action of ASA, including reduction of mutagenesis, inhibition of metastasis and direct inhibition of cell growth (Marnett, 1992). A direct effect of ASA on cell growth of normal human foreskin fibroblasts was ruled out (Lanas et al., 1994). However, indomethacin, a reversible inhibitor of PGHS, arrests human fibroblasts and rat hepatoma cells in the G₁ phase of the cycle (Bayer et al., 1979; Hial et al., 1977) and inhibits the proliferation of Swiss 3T3 cells (Mehmet et al., 1990; Rozengurt et al., 1983).

Swiss 3T3 fibroblasts have been extensively used to analyze the mechanisms of mitogenic stimulation by neuropeptides and polypeptide growth factors. These cells provide a useful model system for the investigation of cell proliferation control (Rozengurt, 1986). The release of arachidonic acid has been implicated as one of the synergistic signals leading to cell proliferation (Gil et al., 1991; Rozengurt, 1991). PDGF and the amphibian tetradecapeptide bombesin are potent mitogen for Swiss 3T3 cells that can stimulate DNA synthesis in the absence of any other growth factor. The effects of these factors are mediated by multiple synergistic signaling pathways, including arachidonic acid release and production of PGE₂ (Domin and Rozengurt, 1992, 1993; Millar and Rozengurt, 1990; Rozengurt et al., 1983). In our study, we analyze the effect of ASA on the mitogenic action of PDGF and bombesin in Swiss 3T3 cells. The results show that ASA inhibits the DNA synthesis induced by these factors and that this effect is mediated through the inhibition of PGHS.

	Methods

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Reagents. Insulin, PDB, PDGF, forskolin, IBMX, EGF, propidium iodide and ASA were obtained from Sigma Chemical Co. (St. Louis, MO). Bombesin was from Peninsula Laboratories. FCS was from Gibco Laboratories (Grand Island, NY). DMEM and Waymouth's medium were from Biological Industries (Kibbutz Bet Haemek, Israel). [³H]Thymidine was purchased from Amersham Corp. (Arlington Heights, IL.). ¹²⁵I-PGE₂ radioimmunoassay system was from New England Nuclear (Boston, MA). All the other reagents were of analytical grade.

Cell culture. Stock cultures of Swiss 3T3 cells were maintained in DMEM supplemented with 10% FCS, L-glutamine (2 mM) penicillin (100 U/ml) and streptomycin (100 µg/ml) in a humidified atmosphere of 10% CO₂, 90% air at 37°C. For experimental purposes 4 × 10⁴ cells were subcultured in 22-mm dishes with 1 ml of DMEM supplemented with 10% FCS and incubated until confluence and quiescence (6-8 days). The quiescence of the cells was confirmed by cytofluorimetric assay of DNA content using an Elite flow cytometer (Coulter Corporation, Miami, FL) after staining with propidium iodide (Vindelov et al., 1983).

[³H]Thymidine incorporation assay. Determinations of DNA synthesis were performed as previously described (Gil et al., 1991). Briefly, quiescent cultures were washed twice with DMEM and incubated in DMEM/Waymouth's medium [1:1(v/v)] containing [³H]thymidine (1 µCi/ml; 1 mM) and various additions. Growth factors and ASA were added at the same time to cultures except in figure 5. After 40 hr, the cultures were washed twice with phosphate-buffered saline and incubated in 5% trichloroacetic acid for 30 min at 4°C. Trichloroacetic acid was then removed and the cultures were washed twice with ethanol and extracted in 0.5 ml of 2% Na₂CO₃, 0.1 M NaOH, 1% sodium dodecyl sulfate. Incorporation was determined by scintillation counting. The results are expressed as the percentage with respect to the maximal response with 10% FCS or as the percentage of inhibition. The [³H]thymidine incorporation in the absence of growth factors was about 40 cpm/µg protein. The number of cells assessed by crystal violet staining (Drysdale et al., 1983) did not decrease significantly after 40 hr of aspirin (1 mM) treatment.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Time-dependence of the inhibition of DNA synthesis by ASA. Quiescent Swiss 3T3 cells were treated with 40 nM bombesin (closed circles) or 0.3 nM PDGF (open circles) and 1 mM ASA was added at the indicated times. The results are expressed as a percentage of the inhibition calculated with respect to the [3H]thymidine incorporation to DNA in the absence of aspirin and are the mean ± S.E.M. of nine determinations from three independent experiments.

Measurement of PGE₂ release. PGE₂ release was determined as described previously (Gil et al., 1991). Quiescent cultures were washed twice with phosphate-buffered saline and incubated at 37°C for the indicated times in the required conditions. The medium was removed and stored at 20°C. All vessels used were made of polypropylene or siliconized glassware. Measurements of PGE₂ were performed by radioimmunoassay using a ¹²⁵I-PGE₂ assay system. Aliquots of sample were diluted in assay buffer containing 0.9% NaCl, 0.01 M EDTA, 0.3% bovine -globulin, 0.005% Triton X-100, 0.05% sodium azide, 25 mM phosphate buffer, pH 6.8. The samples were then bound to a rabbit anti-PGE₂ antibody using ¹²⁵I-PGE₂ as a competitive tracer for 16 hr at 4°C. After this time the immune complexes were precipitated by the addition of 16% polyethylene glycol, 0.05% sodium azide and 50 mM phosphate buffer, pH 6.8 for 30 min at 4°C. Samples were centrifuged for 30 min at 2000 × g and the supernatants removed. The resulting pellets were counted in a gamma counter (Wallac, Turku, Finland). Additions of growth factors to the medium had no effect on the radioimmunoassay.

Data analysis. All data points shown are mean values ± S.E.M. of n separate experiments. Statistical significance of differences was assessed by ANOVA (Fisher PLSD test). Differences between absence and presence of ASA are indicated: * P < .01 and ** P < .001.

	Results

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We first studied the effect of ASA on the stimulation of DNA synthesis by different combinations of mitogenic factors in quiescent Swiss 3T3 fibroblasts (fig. 1). After the protocol described above 90% of cells were in the G₀-G₁ phase of the cell cycle. ASA (1 mM) inhibited the mitogenic action of PDGF and bombesin, which stimulate arachidonic acid release and PGE₂ production in Swiss 3T3 cells (Domin and Rozengurt, 1993; Millar and Rozengurt, 1990). In contrast, ASA had no effect on the DNA synthesis for those combinations of factors that do not activate arachidonic acid release in these cells.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Effect of ASA on DNA synthesis stimulated by different growth factors. Cells were incubated with the growth factors indicated, either in the absence (open bars) or in the presence (solid bars) of 1 mM aspirin. The concentrations used were 0.3 nM PDGF, 40 nM bombesin, 20 nM PDB, 170 µM insulin, 8 nM EGF, 25 µM forskolin and 50 µM IBMX. Values represent the percentage with respect to the maximal response with 10% FCS and are the mean ± S.E.M. of n determinations from three independent experiments. The [3H]thymidine incorporation induced by 10% FCS was about 4000 cpm/µg protein.

To analyze the effect of ASA on bombesin or PDGF-stimulated DNA synthesis, Swiss 3T3 cells were incubated in medium containing progressively increased concentrations of these factors with or without 1 mM ASA. As shown in figure 2, cells treated with 1 mM ASA exhibited a 30 to 40% inhibition on the stimulation of DNA synthesis induced by bombesin at all the mitogenic concentrations tested. The bombesin concentration needed to induce half-maximal stimulation of thymidine incorporation remained unmodified by ASA. The inhibitory effect of ASA was not overcome by saturating concentrations of bombesin.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Dose-response curve for the stimulation of DNA synthesis by bombesin in the absence (open circles) or in the presence (closed circles) of 1 mM ASA. The values represent the percentage with respect to the maximal response with 10% FCS and are the mean ± S.E.M. of nine determinations from three independent experiments.

ASA also modified the dose-dependent stimulation curve of DNA synthesis induced by PDGF (fig. 3). ASA added to cell cultures at subsaturating concentrations of PDGF (0.2-0.4 nM) reduced thymidine incorporation. In the absence of ASA, 0.16 nM PDGF was needed to obtain half-maximal response, whereas in the presence of the drug the concentration needed rose to 0.34 nM. The effect of ASA on PDGF-stimulated mitogenesis can be overcome by increasing PDGF concentration. Thymidine incorporation at saturating PDGF concentrations was not affected by the presence of 1 mM ASA. These results also demonstrate that 1 mM ASA has no toxic effect in Swiss 3T3 cells. The effect of a higher dose of ASA (5 mM) on PDGF-stimulated mitogenesis was studied. Incubation of cells with 5 mM aspirin (table 1) inhibited completely the mitogenic action of saturating concentrations of PDGF, but this treatment also inhibited the mitogenic effect of insulin plus EGF. This effect was not due to cytotoxicity of 5 mM ASA, because the morphology of the cells was not indicative of necrosis or apoptosis and the number of cells decreased only slightly (results not shown).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Dose-response curve for the stimulation of DNA synthesis by PDGF in the absence (open circles) or in the presence (closed circles) of 1 mM ASA. The values represent the percentage with respect to the maximal response with 10% FCS and are the mean ± S.E.M. of three determinations from one representative experiment.

To determine the dose of ASA needed to inhibit DNA synthesis, quiescent Swiss 3T3 cells were treated with 40 nM bombesin or 0.4 nM PDGF and increasing concentrations of ASA. As shown in figure 4, ASA caused a dose-dependent inhibition of thymidine incorporation, the half-maximal effect occurring at approximately 125 µM.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Dose response curve for the inhibition of DNA synthesis by ASA. Cultures of Swiss 3T3 were incubated in medium containing 40 nM bombesin (closed circles) or 0.4 nM PDGF (open circles) and various concentrations of aspirin. DNA synthesis was assessed as described in "Methods." The results are expressed as the percentage of inhibition and are the mean ± S.E.M. of nine determinations from three independent experiments.

We next studied the effects of ASA when this drug was added at different times after mitogenic stimulation. As shown in figure 5, the timings of inhibition of bombesin and PDGF-induced DNA synthesis by ASA in Swiss 3T3 cells were different. In the presence of the 40 nM bombesin the greatest inhibitory effect (45% reduction) was seen when ASA was added during the first hour of incubation, whereas only a slight effect was noted when it was added 2 hr after bombesin. Addition of ASA between 6 and 32 hr had no effect on DNA synthesis (fig. 5 and data not shown). In the presence of 0.3 nM PDGF the maximum effect was observed during the first hour, attaining 60% reduction in [³H]thymidine incorporation, but the effect persisted at 2 hr (50% reduction) and was still evident (33%) after 8 hr.

In Swiss 3T3 cells stimulated by PDGF, the major arachidonic acid metabolite is PGE₂, a product of the PGHS pathway (Domin and Rozengurt, 1993; Rozengurt et al., 1983). The synthesis of PGE₂ in PDGF or bombesin-stimulated cells was studied (fig. 6). PDGF stimulated PGE₂ synthesis at two distinct intervals: an early burst of PGE₂ formation that was complete within 30 min and another increase in PGE₂ synthesis at 6 hr. This time course is in agreement with published data (Habenicht et al., 1985). The induction of PGE₂ synthesis by bombesin takes place mainly between 30 min and 2 hr. PDGF induced much higher levels of PGE₂ than bombesin.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Time-course of PGE2 synthesis in Swiss 3T3 fibroblasts. Cells were stimulated with 0.3 nM PDGF (A) or 40 nM bombesin (B) and PGE2 concentration was determined as described in "Methods." The results are the mean ± S.E.M. of three determinations.

To determine whether inhibition of PGE₂ production is implicated in the antimitogenic effect of ASA, Swiss 3T3 cells were incubated in medium containing exogenous PGE₂. Figure 7 shows that addition of 280 nM PGE₂ counteracted the effect of ASA on PDGF and bombesin-stimulated DNA synthesis. This concentration of PGE₂ did not counteract the inhibitory effect of 5 mM aspirin on PDGF-stimulated mitogenesis (data not shown). The concentrations of PGE₂ achieved after bombesin stimulation did not overcome the effect of 1 mM ASA (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effect of PGE2 on the inhibition of DNA synthesis by ASA. Cultures of Swiss 3T3 were incubated in medium containing 0.4 nM PDGF or 0.4 nM PDGF together with 1 mM ASA in the absence (open bars) or in the presence (solid bars) of 280 nM PGE2. Values represent the percentage respect to the maximal response with 10% FCS and are the mean ± S.E.M. of nine determinations from three independent experiments. Statistically significant differences between absence and presence of PGE2 are indicated: * P < .01 and ** P < .001.

Increase in cyclic AMP levels and activation PKC are two of the signals induced by PGE₂ (Halushka et al., 1989). To test whether blockage of cyclic AMP or PKC pathways plays a role in the inhibition of mitogenesis induced by ASA, we added IBMX plus forskolin, which increase cyclic AMP levels, or phorbol 12,13 dibutyrate, a stimulator of PKC, to the medium. As shown in Figure 8, agents that increase cyclic AMP levels overcame the inhibitory effects of aspirin on DNA synthesis induced by PDGF. Activation of PKC did not significantly modify the effect of aspirin.

View larger version (19K): [in this window] [in a new window]   Fig. 8.   Effect of cyclic AMP and PKC activation on the inhibition of DNA synthesis by ASA. Cultures of Swiss 3T3 cells were incubated in medium containing increasing concentrations of PDGF alone (A), or in the presence of 25 µM forskolin and 50 µM IBMX (B) or 20 nM PDB (C) in the absence (open circles) or in the presence (closed circles) of 1 mM ASA. The results are expressed as a percentage of the response observed at PDGF in the absence of ASA and are the mean ± S.E.M. of three determinations from one representative experiment.

	Discussion

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Our results clearly show that ASA inhibits the mitogenic action of PDGF and bombesin in Swiss 3T3 fibroblasts. One mechanism by which NSAID may affect tumorogenesis could be their influence on the arachidonic acid cascade, the products of which have been implicated in carcinogenesis (Lupulescu, 1978). However, some reports contradict the hypothesis that inhibition of prostaglandin synthesis has a role in the cytostatic action of antiinflammatory drugs (DeMello et al., 1980). Two lines of evidence that we presented indicate that the antiproliferative action of pharmacological doses of ASA is mediated via inhibition of PGHS. First, ASA only inhibits the mitogenic action of growth factors that increase arachidonic acid release, the substrate of PGHS. Second, the antiproliferative effects of ASA are partially reversed by exogenous PGE₂, the major arachidonate derivative via the PGHS pathway in these cells.

The mechanism of the inhibitory action of ASA is different for bombesin and PDGF. ASA decreases the mitogenic effect of saturating bombesin, but only decreases mitogenesis at subsaturating PDGF concentrations. Similar results have been reported for the inhibition of bombesin- and PDGF-stimulated DNA synthesis by indomethacin, a reversible inhibitor of PGHS (Mehmet et al., 1990; Rozengurt et al., 1983). These data suggest that PG are essential for the maximal mitogenic action of bombesin. In contrast, high concentrations of PDGF can generate a signal that overcomes the inhibition by ASA. Signal transduction pathways triggered by PDGF include phosphoinositide-specific phospholipase C, phosphatidylinositol-3-kinase, Ras-Raf-MAPK protein kinase cascade, phosphotyrosine phosphatase SHPTP-2 and Src tyrosine kinase (Malarkey et al., 1995). We believe that potentiation of any of these signaling pathways could be responsible for the mitogenic effect of PDGF in the presence of ASA.

The mitogenic action of saturating concentrations of PDGF is completely inhibited by 5 mM ASA. However, three different arguments indicate that probably this effect is not mediated by PGHS inhibition: 1) 5 mM ASA also inhibits the mitogenic effect of insulin plus EGF which do not induce arachidonic acid release, 2) the inhibitory effect of 5 mM ASA on PDGF stimulated mitogenesis is not counteracted by exogenous PGE₂, 3) the dose-response of ASA for inhibition of DNA synthesis have two-phase with a plateau between 0.5 and 2 mM, suggesting two different mechanisms. PGHS-independent mechanisms have been proposed to explain the inhibition of NF-B by high concentrations of ASA (Kopp and Ghosh, 1994).

There are two PGHS isoenzymes, PGHS-1 and PGHS-2, which differ in their expression regulation and tissue distribution. PGHS-1 is considered to be involved in cell-cell signaling and maintaining tissue homeostasis, whereas PGHS-2 expression occurs in a limited number of cell types and is regulated by specific stimulatory events (Vane, 1994). Growth factors and tumor promoters induce the expression of PGHS-2 in fibroblasts, leading to the hypothesis that PGHS-2 isoform is involved in mitogenesis (Kujubu et al., 1991). Synthesis of PGHS-2 protein increased 2 hr after mitogen activation and the level of PGHS-2 protein peaked between 6 and 8 hr (Kujubu et al., 1993). The effect of ASA on bombesin-stimulated DNA synthesis is mainly produced within the first hour. These time-dependent effects of ASA suggest that PGHS-1 activity is involved in bombesin-stimulated DNA synthesis and that some PGH-derived metabolites produced during the first hour are necessary to obtain the maximal effects of bombesin. The fact that the addition of ASA 8 hr after that of PDGF can inhibit PDGF-stimulated DNA synthesis suggests that both PGHS-1 and PGHS-2 might be involved in the stimulation of DNA synthesis by this factor. The involving of PGHS-2 in PDGF-induced PG production have been demonstrated using antisense PGHS-2 oligonucleotides (Reddy and Herschman, 1994).

The simplest explanation for the effects of ASA is that some PGHS products are involved in DNA synthesis. In Swiss 3T3 cells, the major product of arachidonic acid metabolism is PGE₂ (Domin and Rozengurt, 1993; Habenicht et al., 1985). The timing of the PGE₂ synthesis induced by PDGF or bombesin correlates with the time course of the effect of ASA on [³H]thymidine incorporation. These results also explain why ASA inhibits DNA synthesis when added later than 2 hr after PDGF treatment.

The involvement of PGE₂ in PDGF- and bombesin-stimulated mitogenesis is supported by the finding that the antiproliferative effects of aspirin are counteracted by exogenous PGE₂. The levels of PGE₂ after PDGF treatment are similar to the exogenous PGE₂ concentration that overcome the inhibitory effect of ASA. In contrast, this concentration of exogenous PGE₂ is higher than that induced by bombesin. These results suggest that other products of the PGHS pathway may be involved in the mitogenic action of bombesin.

Two different second messenger systems might be activated after the binding of PGE₂ to different receptor subtypes. The EP2 and EP3 receptors are coupled to adenylate cyclase, although the subtype EP1 stimulates phospholipase C and, subsequently, Ca⁺⁺ mobilization and activation of PKC (Halushka et al., 1989). In Swiss 3T3 cells, some authors have found that PGE₂ increases cyclic AMP (Millar and Rozengurt, 1988; Rozengurt et al., 1983), although others have reported that PGE₂ does not increase cyclic AMP levels significantly and suggested PKC activation (Danesch et al., 1994; Otto et al., 1982). We show that the inhibitory effects of ASA can be overcome by agents that increase cellular cyclic AMP levels rather than by activation of PKC. There are at least two possible explanations for these results: 1) in Swiss 3T3, PGE₂ increases cyclic AMP, and this is the signal transduction pathway inhibited by ASA; 2) cyclic AMP is not the signal inhibited by ASA but it synergizes with the remaining pathways to recover maximal DNA synthesis stimulation.

In addition to its inhibitory effect on DNA synthesis, other actions of NSAID may contribute to the antiproliferative effects of these drugs. Recently, sulindac sulfide and sulindac sulfone have been shown to induce apoptosis in HT-29 human colon carcinoma cells (Piazza et al., 1995; Shiff et al., 1995). Furthermore, different NSAID, but not ASA, cause apoptosis in chicken embryo fibroblasts (Lu et al., 1995). The mechanism responsible for this apoptotic effect of NSAID is not clear, but overexpression of PGHS-2 in rat epithelial intestinal cells inhibits apoptosis (Tsujii and DuBois, 1995).

In conclusion, our data demonstrate that in Swiss 3T3 fibroblasts the inhibition of DNA synthesis by aspirin is mediated through inhibition of PGE₂ synthesis. This antiproliferative action might help to explain the epidemiological data that record a fall in the occurrence of colorectal cancer and other tumors after ASA treatment.