Inhibition of DNA Synthesis by Aspirin in Swiss 3T3 Fibroblasts

0022-3565/97/2801-0366$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics JPET 280:366 ––372, 1997

Vol. 280, No. 1 Printed in U.S.A.

Inhibition of DNA Synthesis by Aspirin in Swiss 3T3 Fibroblasts1 ESTHER CASTANÕ, MIREIA DALMAU, MERCE` MARTI´, FE´LIX BERROCAL, RAMON BARTRONS and JOAN GIL Unitat de Bioquı´mica, Departament de Cie`ncies Fisiolo`giques, Campus de Bellvitge, Universitat de Barcelona, L’Hospitalet (E.C., M.D., R.B., J.G.) and Quı´mica Farmaceútica Bayer S.A. Division Consumer Care. Barcelona, Spain (M.M., F.B.) Accepted for publication September 12, 1996

ABSTRACT In Swiss 3T3 fibroblasts, aspirin inhibited proliferation induced by the complete mitogenic factors platelet-derived growth factor (PDGF) and bombesin. Aspirin decreased the maximum mitogenic effect of bombesin without modifying the concentration necessary to obtain half maximal DNA synthesis stimulation. In contrast, aspirin only decreased mitogenesis at subsaturating PDGF concentrations. The effect of aspirin was found to be concentration dependent. The half-maximal effect occurred at approximately 150 mM. The maximal inhibition was obtained when aspirin was added during the first hour after growth factor addition. At this time, both PDGF and bombesin

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 Received for publication February 14, 1996. 1 This work was supported by grants from “Marato´ de TV3,” the “Fundacio´ August Pi i Sunyer” (Campus de Bellvitge), the “Generalitat de Catalunya” (GRQ93/1131) and Quı´mica Farmaceútica Bayer S.A. (Division Consumer Care).

induced prostaglandin E2 synthesis. PDGF induced much higher levels of prostaglandin E2 than bombesin. The inhibitory effects of aspirin on PDGF or bombesin-stimulated DNA synthesis were counteracted by 280 nM prostaglandin E2. Aspirin effects were overcome by agents that increase cellular cyclic adenosine monophosphate levels but not by activation of protein kinase C. The significance of the antiproliferative action of aspirin might be associated with epidemiological data that show a reduced incidence of colorectal and other cancers after aspirin treatment.

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

ABBREVIATIONS: ASA, aspirin; DMEM, Dulbecco’s modified Eagle’s medium; EGF, epidermal growth factor; FCS, fetal calf serum; IBMX, 3-isobutyl-l-methylxanthine; NSAID, nonsteroidal antiinflammatory drugs; PDB, phorbol 12,13-dibutyrate; PDGF, platelet-derived growth factor; PG, prostaglandin; PGE2, E2-type PG; PGHS, PG synthetase (E.C. 1.1499.1); PKC, protein kinase C; FAP, familiar adenomatous polyposis. 366

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Mitogenesis Inhibition by Aspirin

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 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 mg/ml) in a humidified atmosphere of 10% CO2, 90% air at 37°C. For experimental purposes 4 3 104 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 mCi/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 phosphatebuffered 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/mg 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. 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 220°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 g-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 3 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 6 S.E.M. of n separate experiments. Statistical significance of differences was

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assessed by ANOVA (Fisher PLSD test). Differences between absence and presence of ASA are indicated: * P , .01 and ** P , .001.

Results 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. 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.

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 mM insulin, 8 nM EGF, 25 mM forskolin and 50 mM IBMX. Values represent the percentage with respect to the maximal response with 10% FCS and are the mean 6 S.E.M. of n determinations from three independent experiments. The [3H]thymidine incorporation induced by 10% FCS was about 4000 cpm/mg protein.

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Vol. 280 TABLE 1 Effect of 5 mM aspirin on the DNA synthesisa ASA (5 mM)

3 H-Thymidine Incorporation (%)

PDGF n 5 15

2 1

93 6 2 2 6 0.4

EGF 1 insulin n56

2 1

98 6 5 11 6 7

Addition

a Cells were incubated with the growth factors, either in the absence or in the presence of 5 mM aspirin. The concentrations used were 0.4 nM PDGF, 170 mM insulin and 8 nM EGF. Values represent the percentage with respect to the maximal response with 10% FCS and are the mean 6 S.E.M. of n determinations from at least three independent experiments.

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 6 S.E.M. of nine determinations from three independent experiments.

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). 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 mM. 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

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 6 S.E.M. of three determinations from one representative experiment.

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

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 6 S.E.M. of nine determinations from three independent experiments.

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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 6 S.E.M. of nine determinations from three independent experiments.

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. To determine whether inhibition of PGE2 production is


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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 6 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.

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). 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,

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 6 S.E.M. of three determinations.

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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 mM forskolin and 50 mM 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 6 S.E.M. of three determinations from one representative experiment.

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.

Discussion 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 ex-

ogenous 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 PDGFstimulated 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 Cg, 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. PGHSindependent mechanisms have been proposed to explain the inhibition of NF-kB 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

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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, Ca11 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. Acknowledgments

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