Inhibition of DNA Synthesis by Aspirin in Swiss 3 T 3 Fibroblasts 1

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 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. 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 Received for publication February 14, 1996. 1 This work was supported by grants from “Marató de TV3,” the “Fundació August Pi i Sunyer” (Campus de Bellvitge), the “Generalitat de Catalunya” (GRQ93/1131) and Quı́mica Farmacéutica Bayer S.A. (Division Consumer Care). 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. 0022-3565/97/2801-0366$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 280, No. 1 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 280:366––372, 1997 366 at A PE T Jornals on July 8, 2017 jpet.asjournals. org D ow nladed from 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.