BMC Cancer 2004, 4:49 http://www.biomedcentral.com/1471-2407/4/49 and tamoxifen is considerably more effective than raloxifene [3]. In the dimethylbenzanthracene (DMBA)- induced model in rats, in which tumor growth is predom- inantly dependent on prolactin for growth, tamoxifen and raloxifene show effective anti-tumor action. Tamoxifen and raloxifene have several properties in com- mon; e.g. prevention of tumors in the DMBA induced rat mammary model, maintenance of bone density in the ovariectomized rat and reduction of low density lipopro- tein cholesterol. The partial estrogen agonist activity of tamoxifen on uterine tissue, however, increases the risk of developing endometrial cancer. This does not appear to occur with raloxifene. Tamoxifen stimulates arachidonic acid release from rat liver cells [4]. In this report, I have compared tamoxifen and the raloxifene analog LY117018 for effectiveness at releasing arachidonic acid (AA) from rat liver, rat glial, human colon carcinoma and human breast carcinoma cells and their effects on prostaglandin (PG) I2 production by the rat liver cells. Although both compounds release AA from these cells, LY117018 is less effective. Only tamoxifen stimulates both basal and PGI2 production induced by incubation of rat liver cells with lactacystin in the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). LY117018, however, inhibits the PGI2 production stimulated by tamoxifen. The intracellular release of AA and/or the cellular reac- tions that accompany that release may initiate pathways that prevent tumor growth. The tissue specific effects of tamoxifen and LY117018 may be associated with the AA or with cyclooxygenase (COX) activity and/or one of the many bioactivities resulting from oxygenation and metab- olism of the released AA. Methods The C-9 rat liver and BT-20 human breast carcinoma cells were purchased from the American Type Culture Collec- tion (Manassas, VA, USA) and maintained in MEM sup- plemented with 10% fetal bovine serum. The C-6 rat glial cell line was obtained from Dr. Elaine Y. Lai in the Depart- ment of Biology, Brandeis University and maintained in medium 199. The human colon carcinoma cells (HT-29) were obtained from Dr. Basil Rigas, American Health Foundation, Valhalla, NY and maintained in McCoy's medium. [3H]AA (91.8 Ci/mmol) was purchased from NEN Life Science Products, Inc. (Boston, MA, USA); ICI- 182,780 from Tocris Cookson, Inc. (Ballwin, MO, USA); tamoxifen and 4-OH-tamoxifen were from Sigma Chemi- cal Co. (St. Louis, MO, USA). LY117018 was obtained from Dr. David A. Cox, Eli Lilly and Co. (Indianapolis, IN, USA). Raloxifene was extracted from EVISTA® tablets with dimethylsulfoxide. Two days prior to experiments, the cells were treated with 0.25% trypsin-EDTA and, after addition of minimum essential medium (MEM), medium 199 or McCoy's medium containing 10% fetal bovine serum, the floating cells were seeded onto 35 mm culture dishes. The plating densities varied from 0.1 to 0.5 × 105 cells/35 mm dish. The freshly seeded cultures were incubated for 24-h to allow for cell attachment. After decantation of incubating media, 1.0 ml fresh media (MEM for the rat liver and BT- 20 cells, medium 199 for the rat glial or McCoy's for the HT-29 cells respectively) containing 10% fetal bovine serum and [3H] AA (0.2 µCi/ml) was added and the cells incubated for 24-h. The cells were washed 4 times with MEM and incubated for various periods of time with 1.0 ml of MEM, medium 199 or McCoy's containing 1.0 mg bovine serum albumin (BSA)/ml and different concentra- tions of each test compound. The culture fluids were then decanted, centrifuged at 2000 × g for 10 min, and 200 µl of the supernate counted for radioactivity. Radioactivity recovered in the washes before the 6-h incubation was compared to input radioactivity to calculate the % radio- activity incorporated into the cells [5]. As measured by thin layer chromatography (TLC), of the [3H] released from radiolabelled methylcholanthrene transformed fibroblasts (about 20% after a 3-h stimula- tion by serum), 92% was AA, 4% was PGE2, 0.6% and 1% were PGF2α and phospholipids respectively [6]. When labelled to equilibrium, the [3H] AA had been incorpo- rated into phosphatidylcholine (50%), phosphatidyleth- anolamine (36%), phodphatidylserine (9%) and triglycerides (10%) [7]. Such distribution varied among 12 cell lines [8]. The [3H] AA label released from human colorectal cancer cell lines (HCT116 and SW180) was AA as measured by TLC [9]. For PGI2 production by the rat liver cells, 1.0 ml of MEM supplemented with 10% fetal bovine serum, lacking [3H] AA, was added after the first 24-h incubation. The cells were incubated for another 24-h, washed three times with MEM, then incubated with lactacystin and TPA in the presence and absence of the test compounds in MEM/BSA for various periods of time. The culture fluids were decanted and analyzed for 6-keto-PGF1α, the stable hydro- lytic product of PGI2, by radioimmunoassay [10]. At the sub-confluent cell densities used in these experiments (about 5 × 104 or less per dish) only the major COX prod- uct of rat liver cells (97% 6-keto-PGF1α) could be meas- ured. The other two COX products, PGE2 (2%) and PGF2α (1%) could not be measured. The release of [3 H] AA is presented as a percentage of the radioactivity incorporated by the cells. Except for the time- course experiments, which used duplicate dishes (Fig. 5), three to six culture dishes were used for each experimental Page 2 of 8 (page number not for citation purposes)