BMC Cancer 2004, 4:49 http://www.biomedcentral.com/1471-2407/4/49 trFAaiAmlgouoxrrexifeleiefnea1nese,a4nfr-dOom(LHBY)-(1hAf1yr)7odr0mao1t8xrlyaiv-ttegarlmiacolexcllesifle(lsCn,i-nL9cY)u1sbt1aim7te0ud1la8wteaitdnhdby AA release from (A) rat liver cells (C-9) stimulated by tamoxifen, 4-OH-hydroxy-tamoxifen, LY117018 and raloxifene and (B) from rat glial cells incubated with tamoxifen and LY117018. The cells were incubated for 6-h. The analyses were performed with triplicate dishes. Each bar gives the mean value and the brackets give the SEM. * = sta- tistically significant vs control. The data are representative of several independent experiments with similar results. point. The data are expressed as mean values ± SEM. The data were evaluated statistically by the unpaired Student's t-test. A P value < 0.05 was considered significant. Results AA Release The release of AA after incubation of the rat liver cells with tamoxifen, 4-OH-tamoxifen, LY117018 and raloxifene for 6 h is shown in Fig. 1-A. Tamoxifen is the most effective: it releases AA at 10 µM. 4-OH-tamoxifen, an active metab- olite of tamoxifen [2] is also effective. Raloxifene and LY117018 release AA from the rat liver cells, but are about 5 times less potent (at the doses tested) than tamoxifen. At µM concentrations, all induce apoptosis [11,12]. Tamoxifen, but not 4-OH-tamoxifen, has also been reported to induce apoptosis in p53(-) normal human mammary epithelial cells [13]. Yet, the metabolite binds to the ER with higher affinity than tamoxifen [14,15]. The release of AA by tamoxifen and LY117018 is not unique to the rat liver cells. Both release AA from rat glial cells (Fig. 1-B). Tamoxifen is more effective at releasing AA than is LY117018. The highest level of [3H] AA released into the medium which contains BSA (1 mg/ml) would be 0.25 nM (the specific activity of the [3H] AA is 92 Ci/mmole). Since TLC or HPLC analyses were not done, the concentration of the total released AA could not be quantitatively estimated. aFAniAgdur(LreBYel)e1ah21su7em,0far1no8mco(lAon) hcuarmcainnobmraeacsetllcsa(rHciTno-2m9a) bceylltsa m(BoTx-i2fe0n) AA release from (A) human breast carcinoma cells (BT-20) and (B) human colon carcinoma cells (HT-29) by tamoxifen and LY117,018. The cells were incubated for 6-h. The analy- ses were performed with triplicate dishes. Each bar gives the mean values and the brackets give the SEM. * = statistically significant vs appropriate MEM/BSA control. The data are representative of several independent experiments. However, even treatment of a colorectal cancer cell line with 200 µM AA for 48-h leads to apoptosis [9]. The release of AA is neither species nor tissue specific. Both drugs also release AA from human breast carcinoma (BT-20) and human colon carcinoma (HT-29) cells (Fig. 2-A and 2-B). Again, LY117018 is less effective than tamoxifen. PGI2 production Of the cultured cells studied, the COX mediated meta- bolic profile of AA has been described only in the rat liver cells. Thus, the effects of tamoxifen and LY117018 on PGI2 production by these cells were determined. Tamoxifen stimulates basal PGI2 production as well as that induced by lactacystin in the presence of TPA. LY117018 and raloxifene (Fig. 3-A) are ineffective in stim- ulating basal PGI2 production. LY117018, at a concentra- tion that does not affect PGI2 production (50 µM), inhibits the PGI2 production stimulated by lactacystin in the presence of TPA (Fig. 3-B). The increased PGI2 produc- tion probably reflects some stimulation of both induced and basal COX activity. In mouse neuronal and embry- onic rat mesencephalic cells, COX-2 is induced by lacta- cystin as measured by western and northern blot analyses [16]. Identification of the induced and basal isoforms stimu- lated by tamoxifen in these rat liver cells was deduced Page 3 of 8 (page number not for citation purposes)